Methods and compositions for protein sequencing

ABSTRACT

Aspects of the application provide methods of identifying and sequencing proteins, polypeptides, and amino acids, and compositions useful for the same. In some aspects, the application provides methods of obtaining data during a degradation process of a polypeptide, and outputting a sequence representative of the polypeptide. In some aspects, the application provides amino acid recognition molecules comprising a shielding element that enhances photostability in polypeptide sequencing reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/907,507, filed Sep. 27, 2019, andU.S. Provisional Patent Application No. 62/768,076, filed Nov. 15, 2018,each of which is hereby incorporated by reference in its entirety.

BACKGROUND

Proteomics has emerged as an important and necessary complement togenomics and transcriptomics in the study of biological systems. Theproteomic analysis of an individual organism can provide insights intocellular processes and response patterns, which lead to improveddiagnostic and therapeutic strategies. The complexity surroundingprotein structure, composition, and modification present challenges indetermining large-scale protein sequencing information for a biologicalsample.

SUMMARY

In some aspects, the application provides methods and compositions fordetermining amino acid sequence information from polypeptides (e.g., forsequencing one or more polypeptides). In some embodiments, amino acidsequence information can be determined for single polypeptide molecules.In some embodiments, the relative position of two or more amino acids ina polypeptide is determined, for example for a single polypeptidemolecule. In some embodiments, one or more amino acids of a polypeptideare labeled (e.g., directly or indirectly) and the relative positions ofthe labeled amino acids in the polypeptide is determined.

In some aspects, the application provides methods comprising obtainingdata during a degradation process of a polypeptide. In some embodiments,the methods further comprise analyzing the data to determine portions ofthe data corresponding to amino acids that are sequentially exposed at aterminus of the polypeptide during the degradation process. In someembodiments, the methods further comprise outputting an amino acidsequence representative of the polypeptide. In some embodiments, thedata is indicative of amino acid identity at the terminus of thepolypeptide during the degradation process. In some embodiments, thedata is indicative of a signal produced by one or more amino acidrecognition molecules binding to different types of terminal amino acidsat the terminus during the degradation process. In some embodiments, thedata is indicative of a luminescent signal generated during thedegradation process. In some embodiments, the data is indicative of anelectrical signal generated during the degradation process.

In some embodiments, analyzing the data further comprises detecting aseries of cleavage events and determining the portions of the databetween successive cleavage events. In some embodiments, analyzing thedata further comprises determining a type of amino acid for each of theindividual portions. In some embodiments, each of the individualportions comprises a pulse pattern (e.g., a characteristic pattern), andanalyzing the data further comprises determining a type of amino acidfor one or more of the portions based on its respective pulse pattern.In some embodiments, determining the type of amino acid furthercomprises identifying an amount of time within a portion when the datais above a threshold value and comparing the amount of time to aduration of time for the portion. In some embodiments, determining thetype of amino acid further comprises identifying at least one pulseduration for each of the one or more portions. In some embodiments,determining the type of amino acid further comprises identifying atleast one interpulse duration for each of the one or more portions. Insome embodiments, the amino acid sequence includes a series of aminoacids corresponding to the portions.

In some aspects, the application provides systems comprising at leastone hardware processor, and at least one non-transitorycomputer-readable storage medium storing processor-executableinstructions that, when executed by the at least one hardware processor,cause the at least one hardware processor to perform a method inaccordance with the application. In some aspects, the applicationprovides at least one non-transitory computer-readable storage mediumstoring processor-executable instructions that, when executed by atleast one hardware processor, cause the at least one hardware processorto perform a method in accordance with the application.

In some aspects, the application provides methods of polypeptidesequencing. In some embodiments, the methods comprise contacting asingle polypeptide molecule with one or more terminal amino acidrecognition molecules. In some embodiments, the methods further comprisedetecting a series of signal pulses indicative of association of the oneor more terminal amino acid recognition molecules with successive aminoacids exposed at a terminus of the single polypeptide molecule while itis being degraded, thereby obtaining sequence information about thesingle polypeptide molecule. In some embodiments, the amino acidsequence of most or all of the single polypeptide molecule isdetermined. In some embodiments, the series of signal pulses is a seriesof real-time signal pulses.

In some embodiments, association of the one or more terminal amino acidrecognition molecules with each type of amino acid exposed at theterminus produces a characteristic pattern in the series of signalpulses that is different from other types of amino acids exposed at theterminus. In some embodiments, a signal pulse of the characteristicpattern corresponds to an individual association event between aterminal amino acid recognition molecule and an amino acid exposed atthe terminus. In some embodiments, the characteristic patterncorresponds to a series of reversible terminal amino acid recognitionmolecule binding interactions with the amino acid exposed at theterminus of the single polypeptide molecule. In some embodiments, thecharacteristic pattern is indicative of the amino acid exposed at theterminus of the single polypeptide molecule and an amino acid at acontiguous position (e.g., amino acids of the same type or differenttypes).

In some embodiments, the single polypeptide molecule is degraded by acleaving reagent that removes one or more amino acids from the terminusof the single polypeptide molecule. In some embodiments, the methodsfurther comprise detecting a signal indicative of association of thecleaving reagent with the terminus. In some embodiments, the cleavingreagent comprises a detectable label (e.g., a luminescent label, aconductivity label). In some embodiments, the single polypeptidemolecule is immobilized to a surface. In some embodiments, the singlepolypeptide molecule is immobilized to the surface through a terminalend distal to the terminus to which the one or more terminal amino acidrecognition molecules associate. In some embodiments, the singlepolypeptide molecule is immobilized to the surface through a linker(e.g., a solubilizing linker comprising a biomolecule).

In some aspects, the application provides methods of sequencing apolypeptide comprising contacting a single polypeptide molecule in areaction mixture with a composition comprising one or more terminalamino acid recognition molecules and a cleaving reagent. In someembodiments, the methods further comprise detecting a series of signalpulses indicative of association of the one or more terminal amino acidrecognition molecules with a terminus of the single polypeptide moleculein the presence of the cleaving reagent. In some embodiments, the seriesof signal pulses is indicative of a series of amino acids exposed at theterminus over time as a result of terminal amino acid cleavage by thecleaving reagent.

In some aspects, the application provides methods of sequencing apolypeptide comprising (a) identifying a first amino acid at a terminusof a single polypeptide molecule, (b) removing the first amino acid toexpose a second amino acid at the terminus of the single polypeptidemolecule, and (c) identifying the second amino acid at the terminus ofthe single polypeptide molecule. In some embodiments, (a)-(c) areperformed in a single reaction mixture. In some embodiments, (a)-(c)occur sequentially. In some embodiments, (c) occurs before (a) and (b).In some embodiments, the single reaction mixture comprises one or moreterminal amino acid recognition molecules. In some embodiments, thesingle reaction mixture comprises a cleaving reagent. In someembodiments, the first amino acid is removed by the cleaving reagent. Insome embodiments, the methods further comprise repeating the steps ofremoving and identifying one or more amino acids at the terminus of thesingle polypeptide molecule, thereby determining a sequence (e.g., apartial sequence or a complete sequence) of the single polypeptidemolecule.

In some aspects, the application provides methods of identifying anamino acid of a polypeptide comprising contacting a single polypeptidemolecule with one or more amino acid recognition molecules that bind tothe single polypeptide molecule. In some embodiments, the methodsfurther comprise detecting a series of signal pulses indicative ofassociation of the one or more amino acid recognition molecules with thesingle polypeptide molecule under polypeptide degradation conditions. Insome embodiments, the methods further comprise identifying a first typeof amino acid in the single polypeptide molecule based on a firstcharacteristic pattern in the series of signal pulses.

In some aspects, the application provides methods of identifying aterminal amino acid (e.g., the N-terminal or the C-terminal amino acid)of a polypeptide. In some embodiments, the methods comprise contacting apolypeptide with one or more labeled affinity reagents (e.g., one ormore amino acid recognition molecules) that selectively bind one or moretypes of terminal amino acids at a terminus of the polypeptide. In someembodiments, the methods further comprise identifying a terminal aminoacid at the terminus of the polypeptide by detecting an interaction ofthe polypeptide with the one or more labeled affinity reagents.

In yet other aspects, the application provides methods of polypeptidesequencing by Edman-type degradation reactions. In some embodiments,Edman-type degradation reactions may be performed by contacting apolypeptide with different reaction mixtures for purposes of eitherdetection or cleavage (e.g., as compared to a dynamic sequencingreaction, which can involve detection and cleavage using a singlereaction mixture).

Accordingly, in some aspects, the application provides methods ofdetermining an amino acid sequence of a polypeptide comprising (i)contacting a polypeptide with one or more labeled affinity reagents thatselectively bind one or more types of terminal amino acids at a terminusof the polypeptide. In some embodiments, the methods further comprise(ii) identifying a terminal amino acid (e.g., the N-terminal or theC-terminal amino acid) at the terminus of the polypeptide by detectingan interaction of the polypeptide with the one or more labeled affinityreagents. In some embodiments, the methods further comprise (iii)removing the terminal amino acid. In some embodiments, the methodsfurther comprise (iv) repeating (i)-(iii) one or more times at theterminus of the polypeptide to determine an amino acid sequence of thepolypeptide.

In some embodiments, the methods further comprise, after (i) and before(ii), removing any of the one or more labeled affinity reagents that donot selectively bind the terminal amino acid. In some embodiments, themethods further comprise, after (ii) and before (iii), removing any ofthe one or more labeled affinity reagents that selectively bind theterminal amino acid.

In some embodiments, removing a terminal amino acid (e.g., (iii))comprises modifying the terminal amino acid by contacting the terminalamino acid with an isothiocyanate (e.g., phenyl isothiocyanate), andcontacting the modified terminal amino acid with a protease thatspecifically binds and removes the modified terminal amino acid. In someembodiments cleaving a terminal amino acid (e.g., (iii)) comprisesmodifying the terminal amino acid by contacting the terminal amino acidwith an isothiocyanate, and subjecting the modified terminal amino acidto acidic or basic conditions sufficient to remove the modified terminalamino acid.

In some embodiments, identifying a terminal amino acid comprisesidentifying the terminal amino acid as being one type of the one or moretypes of terminal amino acids to which the one or more labeled affinityreagents bind. In some embodiments, identifying a terminal amino acidcomprises identifying the terminal amino acid as being a type other thanthe one or more types of terminal amino acids to which the one or morelabeled affinity reagents bind.

In some aspects, the application provides amino acid recognitionmolecules comprising a shielding element, e.g., for enhancedphotostability in polypeptide sequencing reactions. In some aspects, theapplication provides an amino acid recognition molecule of Formula (I):

A-(Y)_(n)-D  (I),

wherein: A is an amino acid binding component comprising at least oneamino acid recognition molecule; each instance of Y is a polymer thatforms a covalent or non-covalent linkage group; n is an integer from 1to 10, inclusive; and D is a label component comprising at least onedetectable label. In some embodiments, D is less than 200 Å in diameter.In some embodiments, -(Y)_(n)— is at least 2 nm in length (e.g., atleast 5 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 50nm, or more, in length). In some embodiments, -(Y)_(n)— is between about2 nm and about 200 nm in length (e.g., between about 2 nm and about 100nm, between about 5 nm and about 50 nm, or between about 10 nm and about100 nm in length). In some embodiments, each instance of Y isindependently a biomolecule or a dendritic polymer (e.g., a polyol, adendrimer). In some embodiments, the application provides a compositioncomprising the amino acid recognition molecule of Formula (I). In someembodiments, the amino acid recognition molecule is soluble in thecomposition.

In some aspects, the application provides an amino acid recognitionmolecule of Formula (II):

A-Y¹-D  (II),

wherein: A is an amino acid binding component comprising at least oneamino acid recognition molecule; Y¹ is a nucleic acid or a polypeptide;D is a label component comprising at least one detectable label. In someembodiments, when Y¹ is a nucleic acid, the nucleic acid forms acovalent or non-covalent linkage group. In some embodiments, providedthat when Y¹ is a polypeptide, the polypeptide forms a non-covalentlinkage group characterized by a dissociation constant (K_(D)) of lessthan 50×10⁻⁹ M. In some embodiments, the K_(D) is less than 1×10⁻⁹ M,less than 1×10⁻¹⁰ M, less than 1×10⁻¹¹ M, or less than 1×10⁻¹² M.

In some aspects, the application provides an amino acid recognitionmolecule comprising: a nucleic acid; at least one amino acid recognitionmolecule attached to a first attachment site on the nucleic acid; and atleast one detectable label attached to a second attachment site on thenucleic acid, where the nucleic acid forms a covalent or non-covalentlinkage group between the at least one amino acid recognition moleculeand the at least one detectable label. In some embodiments, the nucleicacid comprises a first oligonucleotide strand. In some embodiments, thenucleic acid further comprises a second oligonucleotide strandhybridized with the first oligonucleotide strand.

In some aspects, the application provides an amino acid recognitionmolecule comprising: a multivalent protein comprising at least twoligand-binding sites; at least one amino acid recognition moleculeattached to the protein through a first ligand moiety bound to a firstligand-binding site on the protein; and at least one detectable labelattached to the protein through a second ligand moiety bound to a secondligand-binding site on the protein. In some embodiments, the multivalentprotein is an avidin protein.

In some embodiments, a shielded amino acid recognition molecule may beused in polypeptide sequencing methods in accordance with theapplication, or any method known in the art. Accordingly, in someaspects, the application provides methods of polypeptide sequencing(e.g., in an Edman-type degradation reaction, in a dynamic sequencingreaction, or other method known in the art) comprising contacting apolypeptide molecule with one or more shielded amino acid recognitionmolecules of the application. For example, in some embodiments, themethods comprise contacting a polypeptide molecule with at least oneamino acid recognition molecule that comprises a shield or shieldingelement in accordance with the application, and detecting association ofthe at least one amino acid recognition molecule with the polypeptidemolecule.

In some aspects, the application provides methods of identifying aprotein of interest in a mixed sample. In some embodiments, the methodscomprise cleaving a mixed protein sample to produce a plurality ofpolypeptide fragments. In some embodiments, the methods further comprisedetermining an amino acid sequence of at least one polypeptide fragmentof the plurality in a method in accordance with the methods of theapplication. In some embodiments, the methods further compriseidentifying a protein of interest in the mixed sample if the amino acidsequence is uniquely identifiable to the protein of interest.

In some embodiments, methods of identifying a protein of interest in amixed sample comprise cleaving a mixed protein sample to produce aplurality of polypeptide fragments. In some embodiments, the methodsfurther comprise labeling one or more types of amino acids in theplurality of polypeptide fragments with one or more differentluminescent labels. In some embodiments, the methods further comprisemeasuring luminescence over time for at least one labeled polypeptide ofthe plurality. In some embodiments, the methods further comprisedetermining an amino acid sequence of the at least one labeledpolypeptide based on the luminescence detected. In some embodiments, themethods further comprise identifying a protein of interest in the mixedsample if the amino acid sequence is uniquely identifiable to theprotein of interest.

Accordingly, in some embodiments, a polypeptide molecule or protein ofinterest to be analyzed in accordance with the application can be of amixed or purified sample. In some embodiments, the polypeptide moleculeor protein of interest is obtained from a biological sample (e.g.,blood, tissue, saliva, urine, or other biological source). In someembodiments, the polypeptide molecule or protein of interest is obtainedfrom a patient sample (e.g., a human sample).

The details of certain embodiments of the invention are set forth in theDetailed Description of Certain Embodiments, as described below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe Definitions, Examples, Figures, and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that, in someinstances, various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings.

When describing embodiments in reference to the drawings, directionreferences (“above,” “below,” “top,” “bottom,” “left,” “right,”“horizontal,” “vertical,” etc.) may be used. Such references areintended merely as an aid to the reader viewing the drawings in a normalorientation. These directional references are not intended to describe apreferred or only orientation of an embodied device. A device may beembodied in other orientations.

As is apparent from the detailed description, the examples depicted inthe figures and further described for the purpose of illustrationthroughout the application describe non-limiting embodiments, and insome cases may simplify certain processes or omit features or steps forthe purpose of clearer illustration.

FIGS. 1A-1B show an example of polypeptide sequencing by detection (FIG.1A) and analysis (FIG. 1B) of single molecule binding interactions.

FIGS. 1C-1E show various examples of labeled affinity reagents andmethods of use in accordance with the application. FIG. 1C depictsexample configurations of labeled affinity reagents, including labeledenzymes and labeled aptamers which selectively bind one or more types ofterminal amino acids. FIG. 1D generically depicts a degradation-basedprocess of polypeptide sequencing using labeled affinity reagents. FIG.1E shows an example of polypeptide sequencing using labeled aptamers byrepeated cycles of terminal amino acid detection, modification, andcleavage.

FIG. 2 shows an example of polypeptide sequencing in real-time usinglabeled exopeptidases that each selectively binds and cleaves adifferent type of terminal amino acid.

FIGS. 3A-3B show examples of polypeptide sequencing in real-time byevaluating binding interactions of terminal and/or internal amino acidswith labeled affinity reagents and a labeled cleaving reagent (e.g., alabeled non-specific exopeptidase). FIG. 3A shows an example ofreal-time sequencing by detecting a series of pulses in a signal output.FIG. 3B schematically depicts a temperature-dependent sequencingprocess.

FIG. 4 shows an example of polypeptide sequencing in real-time byevaluating binding interactions of terminal and internal amino acidswith labeled affinity reagents and a labeled non-specific exopeptidase.

FIGS. 5A-5E show non-limiting examples of affinity reagents labeledthrough a shielding element. FIG. 5A illustrates single-molecule peptidesequencing with an affinity reagent labeled through a conventionalcovalent linkage. FIG. 5B illustrates single-molecule peptide sequencingwith an affinity reagent comprising a shielding element. FIGS. 5C-5Eillustrate various examples of shielding elements in accordance with theapplication.

FIG. 6 shows an example of identifying polypeptides based on a uniquecombination of amino acids detected in a labeled polypeptide.

FIG. 7 shows an example of polypeptide sequencing by detectingluminescence of a labeled polypeptide which is subjected to repeatedcycles of terminal amino acid modification and cleavage.

FIGS. 8A-8C show an example of polypeptide sequencing by processiveenzymatic cleavage of a labeled polypeptide. FIG. 8A shows an example ofsequencing by processive enzymatic cleavage of a labeled polypeptide byan immobilized terminal peptidase. FIG. 8B shows an example ofsequencing by processive enzymatic cleavage of an immobilized labeledpolypeptide by a terminal peptidase. FIG. 8C schematically illustratesan example of a real-time sequencing process performed in accordancewith FIG. 8B.

FIG. 9 schematically illustrates an example of sequencing bycofactor-based FRET using an immobilized ATP-dependent protease,donor-labeled ATP, and acceptor-labeled amino acids of a polypeptidesubstrate.

FIGS. 10A-10C show various examples of preparing samples and sample wellsurfaces for analysis of polypeptides and proteins in accordance withthe application. FIG. 10A generically depicts an example process ofpreparing terminally modified polypeptides from a protein sample. FIG.10B generically depicts an example process of conjugating a solubilizinglinker to a polypeptide. FIG. 10C shows an example schematic of a samplewell having modified surfaces which may be used to promote singlemolecule immobilization to a bottom surface.

FIG. 11 is a diagram of an illustrative sequence data processingpipeline for analyzing data obtained during a polypeptide degradationprocess, in accordance with some embodiments of the technology describedherein.

FIG. 12 is a flow chart of an illustrative process for determining anamino acid sequence of a polypeptide molecule, in accordance with someembodiments of the technology described herein.

FIG. 13 is a flow chart of an illustrative process for determining anamino acid sequence representative of a polypeptide, in accordance withsome embodiments of the technology described herein.

FIG. 14 is a block diagram of an illustrative computer system that maybe used in implementing some embodiments of the technology describedherein.

FIGS. 15A-15C show experimental data for select peptide-linkerconjugates prepared and evaluated for enhanced solubility provided bydifferent solubilizing linkers. FIG. 15A shows example structures ofpeptide-linker conjugates that were synthesized and evaluated. FIG. 15Bshows results from LCMS which demonstrate peptide cleavage at theN-terminus. FIG. 15C shows results from a loading experiment.

FIG. 16 shows a summary of amino acid cleavage activities for selectexopeptidases based on experimental results.

FIGS. 17A-17C show experimental data for a dye/peptide conjugate assayfor detecting and cleaving terminal amino acids. FIG. 17A shows exampleschemes and structures used for performing a dye/peptide conjugateassay. FIG. 17B shows imaging results for peptide-linker conjugateloading into sample wells in an on-chip assay. FIG. 17C shows examplesignal traces which detected peptide-conjugate loading and terminalamino acid cleavage.

FIGS. 18A-18F show experimental data for a FRET dye/peptide conjugateassay for detecting and cleaving terminal amino acids. FIG. 18A showsexample schemes and structures used for performing a FRET dye/peptideconjugate assay. FIG. 18B shows FRET imaging results for different timepoints. FIG. 18C shows cutting efficiency at the different time points.FIG. 18D shows cutting displayed at each of the different time points.FIG. 18E shows additional FRET imaging results for different time pointswith a proline iminopeptidase from Yersinia pestis (yPIP). FIG. 18Fshows FRET imaging results for different time points with anaminopeptidase from Vibrio proteolyticus (VPr).

FIGS. 19A-19H show experimental data for terminal amino aciddiscrimination by a labeled affinity reagent. FIG. 19A shows a crystalstructure of a ClpS2 protein that was labeled for these experiments.FIG. 19B shows single molecule intensity traces which illustrateN-terminal amino acid discrimination by the labeled ClpS2 protein. FIG.19C is a plot showing mean pulse duration for different terminal aminoacids. FIG. 19D is a plot showing mean interpulse duration for differentterminal amino acids. FIG. 19E shows plots further illustratingdiscriminant pulse durations among the different terminal amino acids.

FIGS. 19F, 19G, and 19H show example results from dwell time analysisdemonstrating leucine recognition by a ClpS protein fromThermosynochoccus elongatus (teClpS). FIG. 19I shows example resultsfrom dwell time analysis demonstrating differentiable recognition ofphenylalanine, leucine, tryptophan, and tyrosine by A. tumefaciensClpS1. FIG. 19J shows example results from dwell time analysisdemonstrating leucine recognition by S. elongatus ClpS2. FIGS. 19K-19Lshow example results from dwell time analysis demonstrating prolinerecognition by GID4.

FIGS. 20A-20D show example results from polypeptide sequencing reactionsconducted in real-time using a labeled ClpS2 recognition protein and anaminopeptidase cleaving reagent in the same reaction mixture. FIG. 20Ashows signal trace data for a first sequencing reaction. FIG. 20B showspulse duration statistics for the signal trace data shown in FIG. 20A.FIG. 20C shows signal trace data for a second sequencing reaction. FIG.20D shows pulse duration statistics for the signal trace data shown inFIG. 20C.

FIGS. 21A-21F show experimental data for terminal amino acididentification and cleavage by a labeled exopeptidase. FIG. 21A shows acrystal structure of a proline iminopeptidase (yPIP) that wassite-specifically labeled for these experiments. FIG. 21B shows thedegree of labeling for the purified protein product. FIG. 21C is animage of SDS page confirming site-specific labeling of yPIP. FIG. 21D isan overexposed image of the SDS page gel confirming site-specificlabeling. FIG. 21E is an image of a Coomassie stained gel confirmingpurity of labeled protein product. FIG. 21F is an HPLC tracedemonstrating cleavage activity of the labeled exopeptidase. Thesequence YPYPYPK corresponds to SEQ ID NO: 82. The sequence PYPYPKcorresponds to SEQ ID NO: 83.

FIGS. 22A-22F show data from experiments evaluating recognition of aminoacids containing specific post-translational modifications. FIG. 22Ashows representative traces which demonstrated phospho-tyrosinerecognition by an SH2 domain-containing protein; FIG. 22B shows pulseduration data corresponding to the traces of FIG. 22A; and FIG. 22Cshows statistics determined for the traces. FIGS. 22D-22F showrepresentative traces from negative control experiments.

FIG. 23 is a plot showing median pulse duration from experimentsevaluating the effects of penultimate amino acids on pulse duration.

FIGS. 24A-24C show data from experiments evaluating simultaneous aminoacid recognition by differentially labeled recognition molecules. FIG.24A shows a representative trace. FIG. 24B is a plot comparing pulseduration data obtained during these experiments for each recognitionmolecule. FIG. 24C shows pulse duration statistics for theseexperiments.

FIGS. 25A-25C show data from experiments evaluating the photostabilityof peptides during single-molecule recognition. FIG. 25A shows arepresentative trace from recognition using atClpS2-V1 labeled with adye ˜2 nm from the amino acid binding site. FIG. 25B shows avisualization of the structure of the ClpS2 protein used in theseexperiments. FIG. 25C shows a representative trace from recognitionusing ClpS2 labeled with a dye >10 nm from the amino acid binding sitethrough a DNA/protein linker.

FIGS. 26A-26D show representative traces from polypeptide sequencingreactions conducted in real-time on a complementarymetal-oxide-semiconductor (CMOS) chip using a ClpS2 recognition proteinlabeled through a DNA/streptavidin linker in the presence of anaminopeptidase cleaving reagent.

FIG. 27 shows representative traces from polypeptide sequencingreactions conducted in real-time using atClpS2-V1 recognition proteinlabeled through a DNA/streptavidin linker in the presence of Pyrococcushorikoshii TET aminopeptidase cleaving reagent.

FIGS. 28A-28J show representative trace data from polypeptide sequencingreactions conducted in real-time using multiple types of exopeptidaseswith differential cleavage specificities. FIG. 28A shows arepresentative trace from a reaction performed with hTET exopeptidase,with expanded pulse pattern regions shown in FIG. 28B. The sequenceYAAWAAFADDDWK in FIG. 28A corresponds to SEQ ID NO: 78. FIG. 28C shows arepresentative trace from a reaction performed with both hTET and yPIPexopeptidases, with expanded pulse pattern regions shown in FIG. 28D,and additional representative traces shown in FIG. 28E. The sequenceFYPLPWPDDDYK in FIG. 28C corresponds to SEQ ID NO: 80. FIG. 28F shows arepresentative trace from a further reaction performed with both hTETand yPIP exopeptidases, with expanded pulse pattern regions shown inFIG. 28G, and additional representative traces shown in FIG. 28H. FIG.28I shows a representative trace from a reaction performed with bothPfuTET and yPIP exopeptidases, with expanded pulse pattern regions shownin FIG. 28J. The sequence YPLPWPDDDYK in FIGS. 28F and 28I correspondsto SEQ ID NO: 81.

DETAILED DESCRIPTION

Aspects of the application relate to methods of protein sequencing andidentification, methods of polypeptide sequencing and identification,methods of amino acid identification, and compositions for performingsuch methods.

In some aspects, the application relates to the discovery of polypeptidesequencing techniques which may be implemented using existing analyticinstruments with few or no device modifications. For example, previouspolypeptide sequencing strategies have involved iterative cycling ofdifferent reagent mixtures through a reaction vessel containing apolypeptide being analyzed. Such strategies may require modification ofan existing analytic instrument, such as a nucleic acid sequencinginstrument, which may not be equipped with a flow cell or similarapparatus capable of reagent cycling. The inventors have recognized andappreciated that certain polypeptide sequencing techniques of theapplication do not require iterative reagent cycling, thereby permittingthe use of existing instruments without significant modifications whichmight increase instrument size. Accordingly, in some aspects, theapplication provides methods of polypeptide sequencing that permit theuse of smaller sequencing instruments. In some aspects, the applicationrelates to the discovery of polypeptide sequencing techniques that allowboth genomic and proteomic analyses to be performed using the samesequencing instrument.

The inventors have further recognized and appreciated that differentialbinding interactions can provide an additional or alternative approachto conventional labeling strategies in polypeptide sequencing.Conventional polypeptide sequencing can involve labeling each type ofamino acid with a uniquely identifiable label. This process can belaborious and prone to error, as there are at least twenty differenttypes of naturally occurring amino acids in addition to numerouspost-translational variations thereof. In some aspects, the applicationrelates to the discovery of techniques involving the use of amino acidrecognition molecules which differentially associate with differenttypes of amino acids to produce detectable characteristic signaturesindicative of an amino acid sequence of a polypeptide. Accordingly,aspects of the application provide techniques that do not requirepolypeptide labeling and/or harsh chemical reagents used in certainconventional polypeptide sequencing approaches, thereby increasingthroughput and/or accuracy of sequence information obtained from asample.

In some aspects, the application relates to the discovery that apolypeptide sequencing reaction can be monitored in real-time using onlya single reaction mixture (e.g., without requiring iterative reagentcycling through a reaction vessel). As detailed above, conventionalpolypeptide sequencing reactions can involve exposing a polypeptide todifferent reagent mixtures to cycle between steps of amino aciddetection and amino acid cleavage. Accordingly, in some aspects, theapplication relates to an advancement in next generation sequencing thatallows for the analysis of polypeptides by amino acid detectionthroughout an ongoing degradation reaction in real-time. Approaches forsuch polypeptide analysis by dynamic sequencing are described below.

As described herein, in some aspects, the application provides methodsof sequencing a polypeptide by obtaining data during a polypeptidedegradation process, and analyzing the data to determine portions of thedata corresponding to amino acids that are sequentially exposed at aterminus of the polypeptide during the degradation process. In someembodiments, the portions of the data comprise a series of signal pulsesindicative of association of one or more amino acid recognitionmolecules with successive amino acids exposed at the terminus of thepolypeptide (e.g., during a degradation). In some embodiments, theseries of signal pulses corresponds to a series of reversible singlemolecule binding interactions at the terminus of the polypeptide duringthe degradation process.

A non-limiting example of polypeptide sequencing by detecting singlemolecule binding interactions during a polypeptide degradation processis schematically illustrated in FIG. 1A. An example signal trace (I) isshown with a series of panels (II) that depict different associationevents at times corresponding to changes in the signal. As shown, anassociation event between an amino acid recognition molecule (stippledshape) and an amino acid at the terminus of a polypeptide (shown asbeads-on-a-string) produces a change in magnitude of the signal thatpersists for a duration of time.

Panels (A) and (B) depict different association events between an aminoacid recognition molecule and a first amino acid exposed at the terminusof the polypeptide (e.g., a first terminal amino acid). Each associationevent produces a change in the signal trace (I) characterized by achange in magnitude of the signal that persists for the duration of theassociation event. Accordingly, the time duration between theassociation events of panels (A) and (B) may correspond to a duration oftime within which the polypeptide is not detectably associated with anamino acid recognition molecule.

Panels (C) and (D) depict different association events between an aminoacid recognition molecule and a second amino acid exposed at theterminus of the polypeptide (e.g., a second terminal amino acid). Asdescribed herein, an amino acid that is “exposed” at the terminus of apolypeptide is an amino acid that is still attached to the polypeptideand that becomes the terminal amino acid upon removal of the priorterminal amino acid during degradation (e.g., either alone or along withone or more additional amino acids). Accordingly, the first and secondamino acids of the series of panels (II) provide an illustrative exampleof successive amino acids exposed at the terminus of the polypeptide,where the second amino acid became the terminal amino acid upon removalof the first amino acid.

As generically depicted, the association events of panels (C) and (D)produce changes in the signal trace (I) characterized by changes inmagnitude that persist for time durations that are relatively shorterthan that of panels (A) and (B), and the time duration between theassociation events of panels (C) and (D) is relatively shorter than thatof panels (A) and (B). As described herein, in some embodiments, eitherone or both of these distinctive changes in signal may be used todetermine characteristic patterns in the signal trace (I) which candiscriminate between different types of amino acids. In someembodiments, a transition from one characteristic pattern to another isindicative of amino acid cleavage. As used herein, in some embodiments,amino acid cleavage refers to the removal of at least one amino acidfrom a terminus of a polypeptide (e.g., the removal of at least oneterminal amino acid from the polypeptide). In some embodiments, aminoacid cleavage is determined by inference based on a time durationbetween characteristic patterns. In some embodiments, amino acidcleavage is determined by detecting a change in signal produced byassociation of a labeled cleaving reagent with an amino acid at theterminus of the polypeptide. As amino acids are sequentially cleavedfrom the terminus of the polypeptide during degradation, a series ofchanges in magnitude, or a series of signal pulses, is detected. In someembodiments, signal pulse data can be analyzed as illustrated in FIG.1B.

In some embodiments, signal data can be analyzed to extract signal pulseinformation by applying threshold levels to one or more parameters ofthe signal data. For example, panel (III) depicts a threshold magnitudelevel (“M_(L)”) applied to the signal data of the example signal trace(I). In some embodiments, M_(L) is a minimum difference between a signaldetected at a point in time and a baseline determined for a given set ofdata. In some embodiments, a signal pulse (“sp”) is assigned to eachportion of the data that is indicative of a change in magnitudeexceeding M_(L) and persisting for a duration of time. In someembodiments, a threshold time duration may be applied to a portion ofthe data that satisfies M_(L) to determine whether a signal pulse isassigned to that portion. For example, experimental artifacts may giverise to a change in magnitude exceeding M_(L) that does not persist fora duration of time sufficient to assign a signal pulse with a desiredconfidence (e.g., transient association events which could benon-discriminatory for amino acid type, non-specific detection eventssuch as diffusion into an observation region or reagent sticking withinan observation region). Accordingly, in some embodiments, a signal pulseis extracted from signal data based on a threshold magnitude level and athreshold time duration.

Extracted signal pulse information is shown in panel (III) with theexample signal trace (I) superimposed for illustrative purposes. In someembodiments, a peak in magnitude of a signal pulse is determined byaveraging the magnitude detected over a duration of time that persistsabove M_(L). It should be appreciated that, in some embodiments, a“signal pulse” as used herein can refer to a change in signal data thatpersists for a duration of time above a baseline (e.g., raw signal data,as illustrated by the example signal trace (I)), or to signal pulseinformation extracted therefrom (e.g., processed signal data, asillustrated in panel (IV)).

Panel (IV) shows the signal pulse information extracted from the examplesignal trace (I). In some embodiments, signal pulse information can beanalyzed to identify different types of amino acids in a sequence basedon different characteristic patterns in a series of signal pulses. Forexample, as shown in panel (IV), the signal pulse information isindicative of a first type of amino acid based on a first characteristicpattern (“CP₁”) and a second type of amino acid based on a secondcharacteristic pattern (“CP₂”). By way of example, the two signal pulsesdetected at earlier time points provide information indicative of thefirst amino acid at the terminus of the polypeptide based on CP₁, andthe two signal pulses detected at later time points provide informationindicative of the second amino acid at the terminus of the polypeptidebased on CP₂.

Also as shown in panel (IV), each signal pulse comprises a pulseduration (“pd”) corresponding to an association event between the aminoacid recognition molecule and the amino acid of the characteristicpattern. In some embodiments, the pulse duration is characteristic of adissociation rate of binding. Also as shown, each signal pulse of acharacteristic pattern is separated from another signal pulse of thecharacteristic pattern by an interpulse duration (“ipd”). In someembodiments, the interpulse duration is characteristic of an associationrate of binding. In some embodiments, a change in magnitude (“ΔM”) canbe determined for a signal pulse based on a difference between baselineand the peak of a signal pulse. In some embodiments, a characteristicpattern is determined based on pulse duration. In some embodiments, acharacteristic pattern is determined based on pulse duration andinterpulse duration. In some embodiments, a characteristic pattern isdetermined based on any one or more of pulse duration, interpulseduration, and change in magnitude.

Accordingly, as illustrated by FIGS. 1A-1B, in some embodiments,polypeptide sequencing is performed by detecting a series of signalpulses indicative of association of one or more amino acid recognitionmolecules with successive amino acids exposed at the terminus of apolypeptide in an ongoing degradation reaction. The series of signalpulses can be analyzed to determine characteristic patterns in theseries of signal pulses, and the time course of characteristic patternscan be used to determine an amino acid sequence of the polypeptide.

In some embodiments, the series of signal pulses comprises a series ofchanges in magnitude of an optical signal over time. In someembodiments, the series of changes in the optical signal comprises aseries of changes in luminescence produced during association events. Insome embodiments, luminescence is produced by a detectable labelassociated with one or more reagents of a sequencing reaction. Forexample, in some embodiments, each of the one or more amino acidrecognition molecules comprises a luminescent label. In someembodiments, a cleaving reagent comprises a luminescent label. Examplesof luminescent labels and their use in accordance with the applicationare provided elsewhere herein.

In some embodiments, the series of signal pulses comprises a series ofchanges in magnitude of an electrical signal over time. In someembodiments, the series of changes in the electrical signal comprises aseries of changes in conductance produced during association events. Insome embodiments, conductivity is produced by a detectable labelassociated with one or more reagents of a sequencing reaction. Forexample, in some embodiments, each of the one or more amino acidrecognition molecules comprises a conductivity label. Examples ofconductivity labels and their use in accordance with the application areprovided elsewhere herein. Methods for identifying single moleculesusing conductivity labels have been described (see, e.g., U.S. PatentPublication No. 2017/0037462).

In some embodiments, the series of changes in conductance comprises aseries of changes in conductance through a nanopore. For example,methods of evaluating receptor-ligand interactions using nanopores havebeen described (see, e.g., Thakur, A. K. & Movileanu, L. (2019) NatureBiotechnology 37(1)). The inventors have recognized and appreciated thatsuch nanopores may be used to monitor polypeptide sequencing reactionsin accordance with the application. Accordingly, in some embodiments,the application provides methods of polypeptide sequencing comprisingcontacting a single polypeptide molecule with one or more amino acidrecognition molecules, where the single polypeptide molecule isimmobilized to a nanopore. In some embodiments, the methods furthercomprise detecting a series of changes in conductance through thenanopore indicative of association of the one or more terminal aminoacid recognition molecules with successive amino acids exposed at aterminus of the single polypeptide while the single polypeptide is beingdegraded, thereby sequencing the single polypeptide molecule.

In some aspects, the application provides methods of sequencing and/oridentifying an individual protein in a complex mixture of proteins byidentifying one or more types of amino acids of a polypeptide from themixture. In some embodiments, one or more amino acids (e.g., terminalamino acids and/or internal amino acids) of the polypeptide are labeled(e.g., directly or indirectly, for example using a binding agent such asan amino acid recognition molecule) and the relative positions of thelabeled amino acids in the polypeptide are determined. In someembodiments, the relative positions of amino acids in a polypeptide aredetermined using a series of amino acid labeling and cleavage steps.However, in some embodiments, the relative position of labeled aminoacids in a polypeptide can be determined without removing amino acidsfrom the polypeptide but by translocating a labeled polypeptide througha pore (e.g., a protein channel) and detecting a signal (e.g., a FRETsignal) from the labeled amino acid(s) during translocation through thepore in order to determine the relative position of the labeled aminoacids in the polypeptide molecule.

In some embodiments, the identity of a terminal amino acid (e.g., anN-terminal or a C-terminal amino acid) is assessed after which theterminal amino acid is removed and the identity of the next amino acidat the terminus is assessed, and this process is repeated until aplurality of successive amino acids in the polypeptide are assessed. Insome embodiments, assessing the identity of an amino acid comprisesdetermining the type of amino acid that is present. In some embodiments,determining the type of amino acid comprises determining the actualamino acid identity, for example by determining which of thenaturally-occurring 20 amino acids is the terminal amino acid is (e.g.,using a binding agent that is specific for an individual terminal aminoacid). In some embodiments, the type of amino acid is selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, selenocysteine, serine, threonine,tryptophan, tyrosine, and valine.

However, in some embodiments assessing the identity of a terminal aminoacid type can comprise determining a subset of potential amino acidsthat can be present at the terminus of the polypeptide. In someembodiments, this can be accomplished by determining that an amino acidis not one or more specific amino acids (and therefore could be any ofthe other amino acids). In some embodiments, this can be accomplished bydetermining which of a specified subset of amino acids (e.g., based onsize, charge, hydrophobicity, post-translational modification, bindingproperties) could be at the terminus of the polypeptide (e.g., using abinding agent that binds to a specified subset of two or more terminalamino acids).

In some embodiments, assessing the identity of a terminal amino acidtype comprises determining that an amino acid comprises apost-translational modification. Non-limiting examples ofpost-translational modifications include acetylation, ADP-ribosylation,caspase cleavage, citrullination, formylation, N-linked glycosylation,O-linked glycosylation, hydroxylation, methylation, myristoylation,neddylation, nitration, oxidation, palmitoylation, phosphorylation,prenylation, S-nitrosylation, sulfation, sumoylation, andubiquitination.

In some embodiments, assessing the identity of a terminal amino acidtype comprises determining that an amino acid comprises a side chaincharacterized by one or more biochemical properties. For example, anamino acid may comprise a nonpolar aliphatic side chain, a positivelycharged side chain, a negatively charged side chain, a nonpolar aromaticside chain, or a polar uncharged side chain. Non-limiting examples of anamino acid comprising a nonpolar aliphatic side chain include alanine,glycine, valine, leucine, methionine, and isoleucine. Non-limitingexamples of an amino acid comprising a positively charged side chainincludes lysine, arginine, and histidine. Non-limiting examples of anamino acid comprising a negatively charged side chain include aspartateand glutamate. Non-limiting examples of an amino acid comprising anonpolar, aromatic side chain include phenylalanine, tyrosine, andtryptophan. Non-limiting examples of an amino acid comprising a polaruncharged side chain include serine, threonine, cysteine, proline,asparagine, and glutamine.

In some embodiments, a protein or polypeptide can be digested into aplurality of smaller polypeptides and sequence information can beobtained from one or more of these smaller polypeptides (e.g., using amethod that involves sequentially assessing a terminal amino acid of apolypeptide and removing that amino acid to expose the next amino acidat the terminus).

In some embodiments, a polypeptide is sequenced from its amino (N)terminus. In some embodiments, a polypeptide is sequenced from itscarboxy (C) terminus. In some embodiments, a first terminus (e.g., N orC terminus) of a polypeptide is immobilized and the other terminus(e.g., the C or N terminus) is sequenced as described herein.

As used herein, sequencing a polypeptide refers to determining sequenceinformation for a polypeptide. In some embodiments, this can involvedetermining the identity of each sequential amino acid for a portion (orall) of the polypeptide. However, in some embodiments, this can involveassessing the identity of a subset of amino acids within the polypeptide(e.g., and determining the relative position of one or more amino acidtypes without determining the identity of each amino acid in thepolypeptide). However, in some embodiments amino acid contentinformation can be obtained from a polypeptide without directlydetermining the relative position of different types of amino acids inthe polypeptide. The amino acid content alone may be used to infer theidentity of the polypeptide that is present (e.g., by comparing theamino acid content to a database of polypeptide information anddetermining which polypeptide(s) have the same amino acid content).

In some embodiments, sequence information for a plurality of polypeptideproducts obtained from a longer polypeptide or protein (e.g., viaenzymatic and/or chemical cleavage) can be analyzed to reconstruct orinfer the sequence of the longer polypeptide or protein.

Accordingly, in some embodiments, the one or more types of amino acidsare identified by detecting luminescence of one or more labeled affinityreagents that selectively bind the one or more types of amino acids. Insome embodiments, the one or more types of amino acids are identified bydetecting luminescence of a labeled polypeptide.

The inventors have further recognized and appreciated that thepolypeptide sequencing techniques described herein may involvegenerating novel polypeptide sequencing data, particularly in contrastwith conventional polypeptide sequencing techniques. Thus, conventionaltechniques for analyzing polypeptide sequencing data may not besufficient when applied to the data generated using the polypeptidesequencing techniques described herein.

For example, conventional polypeptide sequencing techniques that involveiterative reagent cycling may generate data associated with individualamino acids of a polypeptide being sequenced. In such instances,analyzing the data generated may simply involve determining which aminoacid is being detected at a particular time because the data beingdetected corresponds to only one amino acid. In contrast, thepolypeptide sequencing techniques described herein may generate dataduring a polypeptide degradation process while multiple amino acids ofthe polypeptide molecule are being detected, resulting in data where itmay be difficult to discern between sections of the data correspondingto different amino acids of the polypeptide. Accordingly, the inventorshave developed new computational techniques for analyzing such datagenerated by the polypeptide sequencing techniques described herein thatinvolve determining sections of the data that correspond to individualamino acids, such as by segmenting the data into portions thatcorrespond to respective amino acid association events. Those sectionsmay be then further analyzed to identify the amino acid being detectedduring those individual sections.

As another example, conventional sequencing techniques that involveusing uniquely identifiable labels for each type of amino acid mayinvolve simply analyzing which label is being detected at a particulartime without taking into consideration any dynamics in how individualamino acids interact with other molecules. In contrast, the polypeptidesequencing techniques described herein generate data indicating howamino acids interact with recognition molecules. As discussed above, thedata may include a series of characteristic patterns corresponding toassociation events between amino acids and their respective recognitionmolecules. Accordingly, the inventors have developed new computationaltechniques for analyzing the characteristic patterns to determine a typeof amino acid corresponding to that portion of the data, allowing for anamino acid sequence of a polypeptide to be determined by analyzing aseries of different characteristic patterns.

Labeled Affinity Reagents and Methods of Use

In some embodiments, methods provided herein comprise contacting apolypeptide with a labeled affinity reagent (also referred to herein asan amino acid recognition molecule, which may or may not comprise alabel) that selectively binds one type of terminal amino acid. As usedherein, in some embodiments, a terminal amino acid may refer to anamino-terminal amino acid of a polypeptide or a carboxy-terminal aminoacid of a polypeptide. In some embodiments, a labeled affinity reagentselectively binds one type of terminal amino acid over other types ofterminal amino acids. In some embodiments, a labeled affinity reagentselectively binds one type of terminal amino acid over an internal aminoacid of the same type. In yet other embodiments, a labeled affinityreagent selectively binds one type of amino acid at any position of apolypeptide, e.g., the same type of amino acid as a terminal amino acidand an internal amino acid.

As used herein, in some embodiments, a type of amino acid refers to oneof the twenty naturally occurring amino acids or a subset of typesthereof. In some embodiments, a type of amino acid refers to a modifiedvariant of one of the twenty naturally occurring amino acids or a subsetof unmodified and/or modified variants thereof. Examples of modifiedamino acid variants include, without limitation,post-translationally-modified variants (e.g., acetylation,ADP-ribosylation, caspase cleavage, citrullination, formylation,N-linked glycosylation, O-linked glycosylation, hydroxylation,methylation, myristoylation, neddylation, nitration, oxidation,palmitoylation, phosphorylation, prenylation, S-nitrosylation,sulfation, sumoylation, and ubiquitination), chemically modifiedvariants, unnatural amino acids, and proteinogenic amino acids such asselenocysteine and pyrrolysine. In some embodiments, a subset of typesof amino acids includes more than one and fewer than twenty amino acidshaving one or more similar biochemical properties. For example, in someembodiments, a type of amino acid refers to one type selected from aminoacids with charged side chains (e.g., positively and/or negativelycharged side chains), amino acids with polar side chains (e.g., polaruncharged side chains), amino acids with nonpolar side chains (e.g.,nonpolar aliphatic and/or aromatic side chains), and amino acids withhydrophobic side chains.

In some embodiments, methods provided herein comprise contacting apolypeptide with one or more labeled affinity reagents that selectivelybind one or more types of terminal amino acids. As an illustrative andnon-limiting example, where four labeled affinity reagents are used in amethod of the application, any one reagent selectively binds one type ofterminal amino acid that is different from another type of amino acid towhich any of the other three selectively binds (e.g., a first reagentbinds a first type, a second reagent binds a second type, a thirdreagent binds a third type, and a fourth reagent binds a fourth type ofterminal amino acid). For the purposes of this discussion, one or morelabeled affinity reagents in the context of a method described hereinmay be alternatively referred to as a set of labeled affinity reagents.

In some embodiments, a set of labeled affinity reagents comprises atleast one and up to six labeled affinity reagents. For example, in someembodiments, a set of labeled affinity reagents comprises one, two,three, four, five, or six labeled affinity reagents. In someembodiments, a set of labeled affinity reagents comprises ten or fewerlabeled affinity reagents. In some embodiments, a set of labeledaffinity reagents comprises eight or fewer labeled affinity reagents. Insome embodiments, a set of labeled affinity reagents comprises six orfewer labeled affinity reagents. In some embodiments, a set of labeledaffinity reagents comprises four or fewer labeled affinity reagents. Insome embodiments, a set of labeled affinity reagents comprises three orfewer labeled affinity reagents. In some embodiments, a set of labeledaffinity reagents comprises two or fewer labeled affinity reagents. Insome embodiments, a set of labeled affinity reagents comprises fourlabeled affinity reagents. In some embodiments, a set of labeledaffinity reagents comprises at least two and up to twenty (e.g., atleast two and up to ten, at least two and up to eight, at least four andup to twenty, at least four and up to ten) labeled affinity reagents. Insome embodiments, a set of labeled affinity reagents comprises more thantwenty (e.g., 20 to 25, 20 to 30) affinity reagents. It should beappreciated, however, that any number of affinity reagents may be usedin accordance with a method of the application to accommodate a desireduse.

In accordance with the application, in some embodiments, one or moretypes of amino acids are identified by detecting luminescence of alabeled affinity reagent (e.g., an amino acid recognition moleculecomprising a luminescent label). In some embodiments, a labeled affinityreagent comprises an affinity reagent that selectively binds one type ofamino acid and a luminescent label having a luminescence that isassociated with the affinity reagent. In this way, the luminescence(e.g., luminescence lifetime, luminescence intensity, and otherluminescence properties described elsewhere herein) may be associatedwith the selective binding of the affinity reagent to identify an aminoacid of a polypeptide. In some embodiments, a plurality of types oflabeled affinity reagents may be used in a method according to theapplication, wherein each type comprises a luminescent label having aluminescence that is uniquely identifiable from among the plurality.Suitable luminescent labels may include luminescent molecules, such asfluorophore dyes, and are described elsewhere herein.

In some embodiments, one or more types of amino acids are identified bydetecting one or more electrical characteristics of a labeled affinityreagent. In some embodiments, a labeled affinity reagent comprises anaffinity reagent that selectively binds one type of amino acid and aconductivity label that is associated with the affinity reagent. In thisway, the one or more electrical characteristics (e.g., charge, currentoscillation color, and other electrical characteristics) may beassociated with the selective binding of the affinity reagent toidentify an amino acid of a polypeptide. In some embodiments, aplurality of types of labeled affinity reagents may be used in a methodaccording to the application, wherein each type comprises a conductivitylabel that produces a change in an electrical signal (e.g., a change inconductance, such as a change in amplitude of conductivity andconductivity transitions of a characteristic pattern) that is uniquelyidentifiable from among the plurality. In some embodiments, theplurality of types of labeled affinity reagents each comprises aconductivity label having a different number of charged groups (e.g., adifferent number of negatively and/or positively charged groups).Accordingly, in some embodiments, a conductivity label is a chargelabel. Examples of charge labels include dendrimers, nanoparticles,nucleic acids and other polymers having multiple charged groups. In someembodiments, a conductivity label is uniquely identifiable by its netcharge (e.g., a net positive charge or a net negative charge), by itscharge density, and/or by its number of charged groups.

In some embodiments, an affinity reagent (e.g., an amino acidrecognition molecule) may be engineered by one skilled in the art usingconventionally known techniques. In some embodiments, desirableproperties may include an ability to bind selectively and with highaffinity to one type of amino acid only when it is located at a terminus(e.g., an N-terminus or a C-terminus) of a polypeptide. In yet otherembodiments, desirable properties may include an ability to bindselectively and with high affinity to one type of amino acid when it islocated at a terminus (e.g., an N-terminus or a C-terminus) of apolypeptide and when it is located at an internal position of thepolypeptide. In some embodiments, desirable properties include anability to bind selectively and with low affinity (e.g., with a K_(D) ofabout 50 nM or higher, for example, between about 50 nM and about 50 μM,between about 100 nM and about 10 μM, between about 500 nM and about 50μM) to more than one type of amino acid. For example, in some aspects,the application provides methods of sequencing by detecting reversiblebinding interactions during a polypeptide degradation process.Advantageously, such methods may be performed using an affinity reagentthat reversibly binds with low affinity to more than one type of aminoacid (e.g., a subset of amino acid types).

As used herein, in some embodiments, the terms “selective” and“specific” (and variations thereof, e.g., selectively, specifically,selectivity, specificity) refer to a preferential binding interaction.For example, in some embodiments, a labeled affinity reagent thatselectively binds one type of amino acid preferentially binds the onetype over another type of amino acid. A selective binding interactionwill discriminate between one type of amino acid (e.g., one type ofterminal amino acid) and other types of amino acids (e.g., other typesof terminal amino acids), typically more than about 10- to 100-fold ormore (e.g., more than about 1,000- or 10,000-fold). Accordingly, itshould be appreciated that a selective binding interaction can refer toany binding interaction that is uniquely identifiable to one type ofamino acid over other types of amino acids. For example, in someaspects, the application provides methods of polypeptide sequencing byobtaining data indicative of association of one or more amino acidrecognition molecules with a polypeptide molecule. In some embodiments,the data comprises a series of signal pulses corresponding to a seriesof reversible amino acid recognition molecule binding interactions withan amino acid of the polypeptide molecule, and the data may be used todetermine the identity of the amino acid. As such, in some embodiments,a “selective” or “specific” binding interaction refers to a detectedbinding interaction that discriminates between one type of amino acidand other types of amino acids.

In some embodiments, a labeled affinity reagent (e.g., an amino acidrecognition molecule) selectively binds one type of amino acid with adissociation constant (K_(D)) of less than about 10⁻⁶ M (e.g., less thanabout 10⁻⁷M, less than about 10⁻⁸ M, less than about 10⁻⁹ M, less thanabout 10⁻¹⁰ M, less than about 10⁻¹¹M, less than about 10⁻¹² M, to aslow as 10⁻¹⁶M) without significantly binding to other types of aminoacids. In some embodiments, a labeled affinity reagent selectively bindsone type of amino acid (e.g., one type of terminal amino acid) with aK_(D) of less than about 100 nM, less than about 50 nM, less than about25 nM, less than about 10 nM, or less than about 1 nM. In someembodiments, a labeled affinity reagent selectively binds one type ofamino acid with a K_(D) of between about 50 nM and about 50 μM (e.g.,between about 50 nM and about 500 nM, between about 50 nM and about 5μM, between about 500 nM and about 50 μM, between about 5 μM and about50 μM, or between about 10 μM and about 50 μM). In some embodiments, alabeled affinity reagent selectively binds one type of amino acid with aK_(D) of about 50 nM.

In some embodiments, a labeled affinity reagent (e.g., an amino acidrecognition molecule) selectively binds two or more types of amino acidswith a dissociation constant (K_(D)) of less than about 10⁻⁶ M (e.g.,less than about 10⁻⁷ M, less than about 10−8 M, less than about 10⁻⁹M,less than about 10⁻¹⁰ M, less than about 10⁻¹¹M, less than about 10⁻¹²M, to as low as 10⁻¹⁶ M). In some embodiments, a labeled affinityreagent selectively binds two or more types of amino acids with a K_(D)of less than about 100 nM, less than about 50 nM, less than about 25 nM,less than about 10 nM, or less than about 1 nM. In some embodiments, alabeled affinity reagent selectively binds two or more types of aminoacids with a K_(D) of between about 50 nM and about 50 μM (e.g., betweenabout 50 nM and about 500 nM, between about 50 nM and about 5 μM,between about 500 nM and about 50 μM, between about 5 μM and about 50μM, or between about 10 μM and about 50 μM). In some embodiments, alabeled affinity reagent selectively binds two or more types of aminoacids with a K_(D) of about 50 nM.

In accordance with the methods and compositions provided herein, FIG. 1Cshows various example configurations and uses of labeled affinityreagents. In some embodiments, a labeled affinity reagent 100 comprisesa luminescent label 110 (e.g., a label) and an affinity reagent (shownas stippled shapes) that selectively binds one or more types of terminalamino acids of a polypeptide 120. In some embodiments, an affinityreagent is selective for one type of amino acid or a subset (e.g., fewerthan the twenty common types of amino acids) of types of amino acids ata terminal position or at both terminal and internal positions.

As described herein, an affinity reagent (also known as a “recognitionmolecule”) may be any biomolecule capable of selectively or specificallybinding one molecule over another molecule (e.g., one type of amino acidover another type of amino acid, as with an “amino acid recognitionmolecule” referred to herein). In some embodiments, an affinity reagentis not a peptidase or does not have peptidase activity. For example, insome embodiments, methods of polypeptide sequencing of the applicationinvolve contacting a polypeptide molecule with one or more affinityreagents and a cleaving reagent. In such embodiments, the one or moreaffinity reagents do not have peptidase activity, and removal of one ormore amino acids from the polypeptide molecule (e.g., amino acid removalfrom a terminus of the polypeptide molecule) is performed by thecleaving reagent.

Affinity reagents (e.g., recognition molecules) include, for example,proteins and nucleic acids, which may be synthetic or recombinant. Insome embodiments, an affinity reagent or recognition molecule may be anantibody or an antigen-binding portion of an antibody, an SH2domain-containing protein or fragment thereof, or an enzymaticbiomolecule, such as a peptidase, an aminotransferase, a ribozyme, anaptazyme, or a tRNA synthetase, including aminoacyl-tRNA synthetases andrelated molecules described in U.S. patent application Ser. No.15/255,433, filed Sep. 2, 2016, titled “MOLECULES AND METHODS FORITERATIVE POLYPEPTIDE ANALYSIS AND PROCESSING.”

In some embodiments, an affinity reagent or recognition molecule of theapplication is a degradation pathway protein. Examples of degradationpathway proteins suitable for use as recognition molecules include,without limitation, N-end rule pathway proteins, such as Arg/N-end rulepathway proteins, Ac/N-end rule pathway proteins, and Pro/N-end rulepathway proteins. In some embodiments, a recognition molecule is anN-end rule pathway protein selected from a Gid protein (e.g., Gid4 orGid10 protein), a UBR box protein (e.g., UBR1, UBR2) or UBR boxdomain-containing protein fragment thereof, a p62 protein or ZZdomain-containing fragment thereof, and a ClpS protein (e.g., ClpS1,ClpS2).

In some embodiments, an affinity reagent or recognition molecule of theapplication is a ClpS protein, such as Agrobacterium tumifaciens ClpS1,Agrobacterium tumifaciens ClpS2, Synechococcus elongatus ClpS1,Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS,Escherichia coli ClpS, or Plasmodium falciparum ClpS. In someembodiments, the recognition molecule is an L/F transferase, such asEscherichia coli leucyl/phenylalanyl-tRNA-protein transferase. In someembodiments, the recognition molecule is a D/E leucyltransferase, suchas Vibrio vulnificus Aspartate/glutamate leucyltransferase Bpt. In someembodiments, the recognition molecule is a UBR protein or UBR-boxdomain, such as the UBR protein or UBR-box domain of human UBR1 and UBR2or Saccharomyces cerevisiae UBR1. In some embodiments, the recognitionmolecule is a p62 protein, such as H. sapiens p62 protein or Rattusnorvegicus p62 protein, or truncation variants thereof that minimallyinclude a ZZ domain. In some embodiments, the recognition molecule is aGid4 protein, such as H. sapiens GID4 or Saccharomyces cerevisiae GID4.In some embodiments, the recognition molecule is a Gid10 protein, suchas Saccharomyces cerevisiae GID10. In some embodiments, the recognitionmolecule is an N-meristoyltransferase, such as Leishmania majorN-meristoyltransferase or H. sapiens N-meristoyltransferase NMT1. Insome embodiments, the recognition molecule is a BIR2 protein, such asDrosophila melanogaster BIR2. In some embodiments, the recognitionmolecule is a tyrosine kinase or SH2 domain of a tyrosine kinase, suchas H. sapiens Fyn SH2 domain, H. sapiens Src tyrosine kinase SH2 domain,or variants thereof, such as H. sapiens Fyn SH2 domain triple mutantsuperbinder. In some embodiments, the recognition molecule is anantibody or antibody fragment, such as a single-chain antibody variablefragment (scFv) against phosphotyrosine or another post-translationallymodified amino acid variant described herein.

Table 1 provides a list of example sequences of amino acid recognitionmolecules. Also shown are the amino acid binding preferences of eachmolecule with respect to amino acid identity at a terminal position of apolypeptide unless otherwise specified in Table 1. It should beappreciated that these sequences and other examples described herein aremeant to be non-limiting, and recognition molecules in accordance withthe application can include any homologs, variants thereof, or fragmentsthereof minimally containing domains or subdomains responsible forpeptide recognition.

TABLE 1 Non-limiting examples of amino acid recognition proteins. SEQBinding ID Name Pref.* NO: Sequence Agrobacterium F, W, Y 1MSDSPVDLKPKPKVKPKLERPKLYKVMLLNDDYTPMSFV tumifaciens ClpS2TVVLKAVFRMSEDTGRRVMMTAHRFGSAVVVVCERDIAE variant 1TKAKEATDLGKEAGFPLMFTTEPEE Agrobacterium F, W, Y 2MSDSPVDLKPKPKVKPKLERPKLYKVMLLNDDYTPREFV tumifaciens ClpS2TVVLKAVFRMSEDTGRRVMMTAHRFGSAVVVVCERDIAE TKAKEATDLGKEAGFPLMFTTEPEEAgrobacterium F, W, Y 3 MSDSPVDLKPKPKVKPKLERPKLYKVMLLNDDYTPREFVtumifaciens ClpS2 TVVLKAVFRMSEDTGRRVMMTAHRFGSAVVVVSERDIAE C71STKAKEATDLGKEAGFPLMFTTEPEE Agrobacterium F, W, Y, L 4MIAEPICMQGEGDGEDGGTNRGTSVITRVKPKTKRPNLY tumifaciens ClpS1RVLLLNDDYTPMEFVIHILERFFQKDREAATRIMLHVHQHGVGECGVFTYEVAETKVSQVMDFARQHQHPLQCVMEKK Agrobacterium F, W, Y 5MSDSPVDLKPKPKVKPKLERPKLYKVMLLNDDYTPMSFV tumifaciens ClpS2TVVLKAVFRMSEDTGRRVMMTAHRFGSAVVVVSERDIAE variant 1 C72STKAKEATDLGKEAGFPLMFTTEPEE Agrobacterium F, W, Y, L 6MIAEPISMQGEGDGEDGGTNRGTSVITRVKPKTKRPNLY tumifaciens ClpS1RVLLLNDDYTPMEFVIHILERFFQKDREAATRIMLHVHQ C7SHGVGECGVFTYEVAETKVSQVMDFARQHQHPLQCVMEKK Agrobacterium F, W, Y, L 7MIAEPISMQGEGDGEDGGTNRGTSVITRVKPKTKRPNLY tumifaciens ClpS1RVLLLNDDYTPMEFVIHILERFFQKDREAATRIMLHVHQ C7SC84SC112SHGVGESGVFTYEVAETKVSQVMDFARQHQHPLQSVMEKK Agrobacterium F, W, Y 8MSDSPVDLKPKPKVKPKLERPKLYKVILLNDDYTPMEFV tumifaciens ClpS2VEVLKRVFNMSEEQARRVMMTAHKKGKAVVGVCPRDIAE thermostableTKAKQATDLAREAGFPLMFTTEPEE variant Agrobacterium F, W, Y 9MSDSPVDLKPKPKVKPKLERPKLYKVILLNDDYTPMEFV tumifaciens ClpS2VEVLKRVFNMSEEQARRVMMTAHKKGKAVVGVSPRDIAE thermostableTKAKQATDLAREAGFPLMFTTEPEE variant C72S Synechococcus F, W, Y 10MAVETIQKPETTTKRKIAPRYRVLLHNDDFNPMEYVVMV elongatus ClpS1LMQTVPSLTQPQAVDIMMEAHTNGTGLVITCDIEPAEFY CEQLKSHGLSSSIEPDD SynechococcusF, W, Y, L, 11 MSPQPDESVLSILGVPRPCVKKRSRNDAFVLTVLTCSLQ elongatus ClpS2V, I AIAAPATAPGTTTTRVRQPYPHFRVIVLDDDVNTFQHVAECLLKYIPGMTGDRAWDLTNQVHYEGAATVWSGPQEQAE LYHEQLRREGLTMAPLEAAThermosynechococcus F, W, Y, L 12MPQERQQVTRKHYPNYKVIVLNDDFNTFQHVAACLMKYI elongatus ClpSPNMTSDRAWELTNQVHYEGQAIVWVGPQEQAELYHEQLL RAGLTMAPLEPE Escherichia coliF, W, Y, L 13 MGKTNDWLDFDQLAEEKVRDALKPPSMYKVILVNDDYTP ClpSMEFVIDVLQKFFSYDVERATQLMLAVHYQGKAICGVFTA EVAETKVAMVNKYARENEHPLLCTLEKAEscherichia coli F, W, Y, L 14 MGKTNDWLDFDQLAEEKVRDALKPPSMYKVILVNDDYTPClpSM40A AEFVIDVLQKFFSYDVERATQLMLAVHYQGKAICGVFTAEVAETKVAMVNKYARENEHPLLCTLEKA Plasmodium F, W, Y, L, 15MFKDLKPFFLCIILLLLLIYKCTHSYNIKNKNCPLNFMN falciparum ClpS ISCVRINNVNKNTNISFPKELQKRPSLVYSQKNFNLEKIKKLRNVIKEIKKDNIKEADEHEKKEREKETSAWKVILYNDDIHNFTYVTDVIVKVVGQISKAKAHTITVEAHSTGQALILSTWKSKAEKYCQELQQNGLTVSIIHESQLKDKQKK Escherichia coli K, R 16MRLVQLSRHSIAFPSPEGALREPNGLLALGGDLSPARLL leucyl/phenylalanylMAYQRGIFPWFSPGDPILWWSPDPRAVLWPESLHISRSM -tRNA-proteinKRFHKRSPYRVTMNYAFGQVIEGCASDREEGTWITRGVV transferaseEAYHRLHELGHAHSIEVWREDELVGGMYGVAQGTLFCGESMFSRMENASKTALLVFCEEFIGHGGKLIDCQVLNDHTASLGACEIPRRDYLNYLNQMRLGRLPNNFWVPRCLFSPQE LE Vibrio vulnificus D, E 17MSSDIHQIKIGLTDNHPCSYLPERKERVAVALEADMHTA Aspartate/glutamateDNYEVLLANGFRRSGNTIYKPHCDSCHSCQPIRISVPDI leucyltransferaseELSRSQKRLLAKARSLSWSMKRNMDENWFDLYSRYIVAR BptHRNGTMYPPKKDDFAHFSRNQWLTTQFLHIYEGQRLIAVAVTDIMDHCASAFYTFFEPEHELSLGTLAVLFQLEFCQEEKKQWLYLGYQIDECPAMNYKVRFHRHQKLVNQRWQ Saccharomyces K, R, H 18MGSVHKHTGRNCGRKFKIGEPLYRCHECGCDDTCVLCIH cerevisiae UBR1CFNPKDHVNHHVCTDICTEFTSGICDCGDEEAWNSPLHC KAEEQ H. sapiens GID4 P 19MSGSKFRGHQKSKGNSYDVEVVLQHVDTGNSYLCGYLKIKGLTEEYPTLTTFFEGEIISKKHPFLTRKWDADEDVDRKHWGKFLAFYQYAKSFNSDDFDYEELKNGDYVFMRWKEQFLVPDHTIKDISGASFAGFYYICFQKSAASIEGYYYHRSS EWYQSLNLTHV Saccharomyces P 20MINNPKVDSVAEKPKAVTSKQSEQAASPEPTPAPPVSRN cerevisiae GID4QYPITFNLTSTAPFHLHDRHRYLQEQDLYKCASRDSLSSLQQLAHTPNGSTRKKYIVEDQSPYSSENPVIVTSSYNHTVCTNYLRPRMQFTGYQISGYKRYQVTVNLKTVDLPKKDCTSLSPHLSGFLSIRGLTNQHPEISTYFEAYAVNHKELGFLSSSWKDEPVLNEFKATDQTDLEHWINFPSFRQLFLMSQKNGLNSTDDNGTTNAAKKLPPQQLPTTPSADAGNISRIFSQEKQFDNYLNERFIFMKWKEKFLVPDALLMEGVDGASYDGFYYIVHDQVTGNIQGFYYHQDAEKFQQLELVPSLKNK VESSDCSFEFA Single-chainphospho-Y 21 MMEVQLQQSGPELVKPGASVMISCRTSAYTFTENTVHWV antibody variableKQSHGESLEWIGGINPYYGGSIFSPKFKGKATLTVDKSS fragment (scFv)STAYMELRSLTSEDSAVYYCARRAGAYYFDYWGQGTTLT againstVSSGGGSGGGSGGGSENVLTQSPAIMSASPGEKVTMTCR phosphotyrosine**ASSSVSSSYLHWYRQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYRTFGGGT KLEIKR H. sapiens Fyn phospho-Y22 MGAMDSIQAEEWYEGKLGRKDAERQLLSFGNPRGTFLIR SH2 domain**ESETTKGAYSLSIRDWDDMKGDHVKHYKIRKLDNGGYYITTRAQFETLQQLVQHYSERAAGLSSRLVVPSHK H. sapiens Fyn phospho-Y 23MGAMDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTFLIR SH2 domain tripleESETVKGAYALSIRDWDDMKGDHVKHYLIRKLDNGGYYI mutantTTRAQFETLQQLVQHYSERAAGLSSRLVVPSHK superbinder** H. sapiens Src phospho-Y24 MGAMDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVR tyrosine kinaseESETTKGAYSLSVSDFDNAKGLNVKHYKIRKLDSGGFYI SH2 domain**TSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSK H. sapiens Src phospho-Y 25MGAMDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVR tyrosine kinaseESEVTKGAYALSVSDFDNAKGLNVKHYLIRKLDSGGFYI SH2 domain tripleTSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSK mutant** H. sapiens p62 K, R, H, 26MASLTVKAYLLGKEDAAREIRRFSFCCSPEPEAEAEAAA fragment 1-310 W, F, YGPGPCERLLSRVAALFPALRPGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEKKECRRDHRPPCAQEAPRNMVHPNVICDGCNGPVVGTRYKCSVCPDYDLCSVCEGKGLHRGHTKLAFPSPFGHLSEGFSHSRWLRKVKHGHFGWPGWEMGPPGNWSPRPPRAGEARPGPTAESASGPSEDPSVNFLKNVGESVAAALSPLGIEVDIDVEHGGKRSRLTPVSPESSSTEEKSSSQPSSCCSDPSKPGGNVEGATQSLAEQ H. sapiens p62 K, R, H, 27MASLTVKAYLLGKEDAAREIRRFSFCCSPEPEAEAEAAA fragment 1-180 W, F, YGPGPCERLLSRVAALFPALRPGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEKKECRRDHRPPCAQEAPRNMVHPNVICDGCNGPVVGTRYKCSVCPDYDLCSVCEGKGLHRGHTKLAFPSPFGHLSEGFSHSRWLRKVKHGHFGWPGWEMGPPGNWSPRPPRAGEARPGPTAESASGPSEDPSVNFLKNVGESVAAALSPLGIEVDIDVEHGGKRSRLTPVSPESSSTEEKSSSQPSSCCSDPSKPGGNVEGATQSLAEQ H. sapiens p62 K, R, H, 28MASLTVKAYLLGKEDAAREIRRFSFCCSPEPEAEAEAAA fragment 126-180 W, F, YGPGPCERLLSRVAALFPALRPGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEKKECRRDHRPPCAQEAPRNMVHPNVICDGCNGPVVGTRYKCSVCPDYDLCSVCEGKGLHRGHTKLAFPSPFGHLSEGFSHSRWLRKVKHGHFGWPGWEMGPPGNWSPRPPRAGEARPGPTAESASGPSEDPSVNFLKNVGESVAAALSPLGIEVDIDVEHGGKRSRLTPVSPESSSTEEKSSSQPSSCCSDPSKPGGNVEGATQSLAEQ H. sapiens p62 K, R, H, 29MASLTVKAYLLGKEDAAREIRRFSFCCSPEPEAEAEAAA protein W, F, YGPGPCERLLSRVAALFPALRPGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEKKECRRDHRPPCAQEAPRNMVHPNVICDGCNGPVVGTRYKCSVCPDYDLCSVCEGKGLHRGHTKLAFPSPFGHLSEGFSHSRWLRKVKHGHFGWPGWEMGPPGNWSPRPPRAGEARPGPTAESASGPSEDPSVNFLKNVGESVAAALSPLGIEVDIDVEHGGKRSRLTPVSPESSSTEEKSSSQPSSCCSDPSKPGGNVEGATQSLAEQMRKIALESEGRPEEQMESDNCSGGDDDWTHLSSKEVDPSTGELQSLQMPESEGPSSLDPSQEGPTGLKEAALYPHLPPEADPRLIESLSQMLSMGFSDEGGWLTRLLQTKNYDIGAALD TIQYSKHPPPL Rattus norvegicusK, R, H, 30 MASLTVKAYLLGKEEAAREIRRFSFCFSPEPEAEAAAGP p62 protein W, F, YGPCERLLSRVAVLFPALRPGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEKKECRREHRPPCAQEARSMVHPNVICDGCNGPVVGTRYKCSVCPDYDLCSVCEGKGLHREHSKLIFPNPFGHLSDSFSHSRWLRKLKHGHFGWPGWEMGPPGNWSPRPPRAGDGRPCPTAESASAPSEDPNVNFLKNVGESVAAALSPLGIEVDIDVEHGGKRSRLTPTSAESSSTGTEDKSGTQPSSCSSEVSKPDGAGEGPAQSLTEQMKKIALESVGQPEELMESDNCSGGDDDWTHLSSKEVDPSTGELQSLQMPESEGPSSLDPSQEGPTGLKEAALYPHLPPEADPRLIESLSQMLSMGFSDEGGWLTRLLQTKNYDIGAALDT IQYSKHPPPL Saccharomyces P, M, V31 MTSLNIMGRKFILERAKRNDNIEEIYTSAYVSLPSSTDT cerevisiae GID10RLPHFKAKEEDCDVYEEGTNLVGKNAKYTYRSLGRHLDFLRPGLRFGGSQSSKYTYYTVEVKIDTVNLPLYKDSRSLDPHVTGTFTIKNLTPVLDKVVTLFEGYVINYNQFPLCSLHWPAEETLDPYMAQRESDCSHWKRFGHFGSDNWSLTERNFGQYNHESAEFMNQRYTYLKWKERFLLDDEEQENQMLDDNHHLEGASFEGFYYVCLDQLTGSVEGYYYHPACELFQKLE LVPTNCDALNTYSSGFEIAUBR-box domain K, R, H 32 MGPLGSLCGRVFKSGETTYSCRDCAIDPTCVLCMDCFQDfrom Homo sapiens SVHKNHRYKMHTSTGGGFCDCGDTEAWKTGPFCVNHEP UBR1UBR-box domain K, R, H 33 MGPLGSLCGRVFKVGEPTYSCRDCAVDPTCVLCMECFLGfrom Homo sapiens SIHRDHRYRMTTSGGGGFCDCGDTEAWKEGPYCQKHE UBR2Leishmania major G 34 MSRNPSNSDAAHAFWSTQPVPQTEDETEKIVFAGPMDEP N-KTVADIPEEPYPIASTFEWWTPNMEAADDIHAIYELLRD meristoyltransferaseNYVEDDDSMFRFNYSEEFLQWALCPPNYIPDWHVAVRRKADKKLLAFIAGVPVTLRMGTPKYMKVKAQEKGEGEEAAKYDEPRHICEINFLCVHKQLREKRLAPILIKEATRRVNRTNVWQAVYTAGVLLPTPYASGQYFHRSLNPEKLVEIRFSGIPAQYQKFQNPMAMLKRNYQLPSAPKNSGLREMKPSDVPQVRRILMNYLDSFDVGPVFSDAEISHYLLPRDGVVFTYVVENDKKVTDFFSFYRIPSTVIGNSNYNLLNAAYVHYYAATSIPLHQLILDLLIVAHSRGFDVCNMVEILDNRSFVEQL KFGAGDGHLRYYFYNWAYPKIKPSQVALVMLH. sapiens N- G 35 MADESETAVKPPAPPLPQMMEGNGNGHEHCSDCENEEDNmeristoyltransferase SYNRGGLSPANDTGAKKKKKKQKKKKEKGSETDSAQDQP NMT1VKMNSLPAERIQEIQKAIELFSVGQGPAKTMEEASKRSYQFWDTQPVPKLGEVVNTHGPVEPDKDNIRQEPYTLPQGFTWDALDLGDRGVLKELYTLLNENYVEDDDNMFRFDYSPEFLLWALRPPGWLPQWHCGVRVVSSRKLVGFISAIPANTHIYDTEKKMVEINFLCVHKKLRSKRVAPVLIREITRRVHLEGIFQAVYTAGVVLPKPVGTCRYWHRSLNPRKLIEVKFSHLSRNMTMQRTMKLYRLPETPKTAGLRPMETKDIPVVHQLLTRYLKQFHLTPVMSQEEVEHWFYPQENIIDTFVVENANGEVTDFLSFYTLPSTIMNHPTHKSLKAAYSFYNVHTQTPLLDLMSDALVLAKMKGFDVFNALDLMENKTFLEKLKFG IGDGNLQYYLYNWKCPSMGAEKVGLVLQDrosophila A 36 MGDVQPETCRPSAASGNYFPQYPEYAIETARLRTFEAWPmelanogaster BIR2 RNLKQKPHQLAEAGFFYTGVGDRVRCFSCGGGLMDWNDNDEPWEQHALWLSQCRFVKLMKGQLYIDTVAAKPVLAEEK EESTSIGGDT *Binding preferencesare inferred from published scientific literature and/or furtherdemonstrated by the inventors in single-molecule experiments, asdescribed herein. **Binding to phosphotyrosine may occur at a peptideterminus or at an internal position.

Accordingly, in some embodiments, the application provides an amino acidrecognition molecule having an amino acid sequence selected from Table 1(or having an amino acid sequence that has at least 50%, at least 60%,at least 70%, at least 80%, 80-90%, 90-95%, 95-99%, or higher, aminoacid sequence identity to an amino acid sequence selected from Table 1).In some embodiments, an amino acid recognition molecule has 25-50%,50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-99%, or higher, amino acidsequence identity to an amino acid recognition molecule listed inTable 1. In some embodiments, an amino acid recognition molecule is amodified amino acid recognition molecule and includes one or more aminoacid mutations relative to a sequence set forth in Table 1.

In some embodiments, an amino acid recognition molecule comprises a tagsequence that provides one or more functions other than amino acidbinding. For example, in some embodiments, a tag sequence comprises abiotin ligase recognition sequence that permits biotinylation of therecognition molecule (e.g., incorporation of one or more biotinmolecules, including biotin and bis-biotin moieties). Additionalexamples of functional sequences in a tag sequence include purificationtags, cleavage sites, and other moieties useful for purification and/ormodification of recognition molecules. Table 2 provides a list ofnon-limiting sequences of terminal tag sequences, any one or more ofwhich may be used in combination with any one of the amino acidrecognition molecules of the application (e.g., in combination with asequence set forth in Table 1). It should be appreciated that the tagsequences shown in Table 2 are meant to be non-limiting, and recognitionmolecules in accordance with the application can include any one or moreof the tag sequences (e.g., His-tags and/or biotinylation tags) at theN- or C-terminus of a recognition molecule polypeptide, split betweenthe N- and C-terminus, or otherwise rearranged as practiced in the art.

TABLE 2 Non-limiting examples of terminal tag sequences. SEQ Name ID NO:Sequence Biotinylation tag 37 GGGSGGGSGGGSGLNDFFEAQKIEWHEBis-biotinylation tag 38 GGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE Bis-biotinylation tag 39GSGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDF FEAQKIEWHEHis/biotinylation tag 40 GHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHEHis/bis-biotinylation 41 GHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGStag GGGSGLNDFFEAQKIEWHE His/bis-biotinylation 42GGSHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGG tag GSGGGSGLNDFFEAQKIEWHEHis/bis-biotinylation 43 GSHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGtag SGGGSGLNDFFEAQKIEWHE Bis-biotinylation/His 44GGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFE tag AQKIEWHEGHHHHHH

In some embodiments, a recognition molecule or affinity reagent of theapplication is a peptidase. A peptidase, also referred to as a proteaseor proteinase, is an enzyme that catalyzes the hydrolysis of a peptidebond. Peptidases digest polypeptides into shorter fragments and may begenerally classified into endopeptidases and exopeptidases, which cleavea polypeptide chain internally and terminally, respectively. In someembodiments, labeled affinity reagent 100 comprises a peptidase that hasbeen modified to inactivate exopeptidase or endopeptidase activity. Inthis way, labeled affinity reagent 100 selectively binds without alsocleaving the amino acid from a polypeptide. In yet other embodiments, apeptidase that has not been modified to inactivate exopeptidase orendopeptidase activity may be used. For example, in some embodiments, alabeled affinity reagent comprises a labeled exopeptidase 102.

In accordance with certain embodiments of the application, proteinsequencing methods may comprise iterative detection and cleavage at aterminal end of a polypeptide. In some embodiments, labeled exopeptidase102 may be used as a single reagent that performs both steps ofdetection and cleavage of an amino acid. As generically depicted, insome embodiments, labeled exopeptidase 102 has aminopeptidase orcarboxypeptidase activity such that it selectively binds and cleaves anN-terminal or C-terminal amino acid, respectively, from a polypeptide.It should be appreciated that, in certain embodiments, labeledexopeptidase 102 may be catalytically inactivated by one skilled in theart such that labeled exopeptidase 102 retains selective bindingproperties for use as a non-cleaving labeled affinity reagent 100, asdescribed herein.

An exopeptidase generally requires a polypeptide substrate to compriseat least one of a free amino group at its amino-terminus or a freecarboxyl group at its carboxy-terminus. In some embodiments, anexopeptidase in accordance with the application hydrolyses a bond at ornear a terminus of a polypeptide. In some embodiments, an exopeptidasehydrolyses a bond not more than three residues from a polypeptideterminus. For example, in some embodiments, a single hydrolysis reactioncatalyzed by an exopeptidase cleaves a single amino acid, a dipeptide,or a tripeptide from a polypeptide terminal end.

In some embodiments, an exopeptidase in accordance with the applicationis an aminopeptidase or a carboxypeptidase, which cleaves a single aminoacid from an amino- or a carboxy-terminus, respectively. In someembodiments, an exopeptidase in accordance with the application is adipeptidyl-peptidase or a peptidyl-dipeptidase, which cleave a dipeptidefrom an amino- or a carboxy-terminus, respectively. In yet otherembodiments, an exopeptidase in accordance with the application is atripeptidyl-peptidase, which cleaves a tripeptide from anamino-terminus. Peptidase classification and activities of each class orsubclass thereof is well known and described in the literature (see,e.g., Gurupriya, V. S. & Roy, S. C. Proteases and Protease Inhibitors inMale Reproduction. Proteases in Physiology and Pathology 195-216 (2017);and Brix, K. & Stocker, W. Proteases: Structure and Function. Chapter1). In some embodiments, a peptidase in accordance with the applicationremoves more than three amino acids from a polypeptide terminus.Accordingly, in some embodiments, the peptidase is an endopeptidase,e.g., that cleaves preferentially at particular positions (e.g., beforeor after a particular amino acid). In some embodiments, the size of apolypeptide cleavage product of endopeptidase activity will will dependon the distribution of cleavage sites (e.g., amino acids) within thepolypeptide being analyzed.

An exopeptidase in accordance with the application may be selected orengineered based on the directionality of a sequencing reaction. Forexample, in embodiments of sequencing from an amino-terminus to acarboxy-terminus of a polypeptide, an exopeptidase comprisesaminopeptidase activity. Conversely, in embodiments of sequencing from acarboxy-terminus to an amino-terminus of a polypeptide, an exopeptidasecomprises carboxypeptidase activity. Examples of carboxypeptidases thatrecognize specific carboxy-terminal amino acids, which may be used aslabeled exopeptidases or inactivated to be used as non-cleaving labeledaffinity reagents described herein, have been described in theliterature (see, e.g., Garcia-Guerrero, M. C., et al. (2018) PNAS115(17)).

Suitable peptidases for use as cleaving reagents and/or affinityreagents (e.g., recognition molecules) include aminopeptidases thatselectively bind one or more types of amino acids. In some embodiments,an aminopeptidase recognition molecule is modified to inactivateaminopeptidase activity. In some embodiments, an aminopeptidase cleavingreagent is non-specific such that it cleaves most or all types of aminoacids from a terminal end of a polypeptide. In some embodiments, anaminopeptidase cleaving reagent is more efficient at cleaving one ormore types of amino acids from a terminal end of a polypeptide ascompared to other types of amino acids at the terminal end of thepolypeptide. For example, an aminopeptidase in accordance with theapplication specifically cleaves alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline,selenocysteine, serine, threonine, tryptophan, tyrosine, and/or valine.In some embodiments, an aminopeptidase is a proline aminopeptidase. Insome embodiments, an aminopeptidase is a proline iminopeptidase. In someembodiments, an aminopeptidase is a glutamate/aspartate-specificaminopeptidase. In some embodiments, an aminopeptidase is amethionine-specific aminopeptidase. In some embodiments, anaminopeptidase is an aminopeptidase set forth in Table 3. In someembodiments, an aminopeptidase cleaving reagent cleaves a peptidesubstrate as set forth in Table 3.

In some embodiments, an aminopeptidase is a non-specific aminopeptidase.In some embodiments, a non-specific aminopeptidase is a zincmetalloprotease. In some embodiments, a non-specific aminopeptidase isan aminopeptidase set forth in Table 4. In some embodiments, anon-specific aminopeptidase cleaves a peptide substrate as set forth inTable 4.

Accordingly, in some embodiments, the application provides anaminopeptidase (e.g., an aminopeptidase recognition molecule, anaminopeptidase cleaving reagent) having an amino acid sequence selectedfrom Table 3 or Table 4 (or having an amino acid sequence that has atleast 50%, at least 60%, at least 70%, at least 80%, 80-90%, 90-95%,95-99%, or higher, amino acid sequence identity to an amino acidsequence selected from Table 3 or Table 4). In some embodiments, anaminopeptidase has 25-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or95-99%, or higher, amino acid sequence identity to an aminopeptidaselisted in Table 3 or Table 4. In some embodiments, an aminopeptidase isa modified aminopeptidase and includes one or more amino acid mutationsrelative to a sequence set forth in Table 3 or Table 4.

TABLE 3 Non-limiting examples of aminopeptidases. SEQ Name ID NO:Sequence L. pneumophila M1 45MMVKQGVFMKTDQSKVKKLSDYKSLDYFVIHVDLQIDLSKKPVESK AminopeptidaseARLTVVPNLNVDSHSNDLVLDGENMTLVSLQMNDNLLKENEYELTK (Glu/Asp Specific)DSLIIKNIPQNTPFTIEMTSLLGENTDLFGLYETEGVALVKAESEGLRRVFYLPDRPDNLATYKTTIIANQEDYPVLLSNGVLIEKKELPLGLHSVTWLDDVPKPSYLFALVAGNLQRSVTYYQTKSGRELPIEFYVPPSATSKCDFAKEVLKEAMAWDERTFNLECALRQHMVAGVDKYASGASEPTGLNLFNTENLFASPETKTDLGILRVLEVVAHEFFHYWSGDRVTIRDWFNLPLKEGLITFRAAMFREELFGTDLIRLLDGKNLDERAPRQSAYTAVRSLYTAAAYEKSADIFRMMMLFIGKEPFIEAVAKFFKDNDGGAVTLEDFIESISNSSGKDLRSFLSWFTESGIPELIVTDELNPDTKQYFLKIKTVNGRNRPIPILMGLLDSSGAEIVADKLLIVDQEEIEFQFENIQTRPIPSLLRSFSAPVHMKYEYSYQDLLLLMQFDTNLYNRCEAAKQLISALINDFCIGKKIELSPQFFAVYKALLSDNSLNEWMLAELITLPSLEELIENQDKPDFEKLNEGRQLIQNALANELKTDFYNLLFRIQISGDDDKQKLKGFDLKQAGLRRLKSVCFSYLLNVDFEKTKEKLILQFEDALGKNMTETALALSMLCEINCEEADVALEDYYHYWKNDPGAVNNWFSIQALAHSPDVIERVKKLMRHGDFDLSNPNKVYALLGSFIKNPFGFHSVTGEGYQLVADAIFDLDKINPTLAANLTEKFTYWDKYDVNRQAMMISTLKIIYSNATSSDVRTMAKKGLDKVKEDLPLPIHLTFHGGSTMQDRTAQLIADGNKENAYQLH E. coli methionine 46MGTAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRI aminopeptidaseCNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKD (Met specific)GDIVNIDVIVIKDGEHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGHGIGRGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLRKDDTIPAIISHD M. smegmatis Proline 47MGTLEANTNGPGSMLSRMPVSSRTVPFGDHETWVQVTTPENAQPHA iminopeptidaseLPLIVLHGGPGMAHNYVANIAALADETGRTVIHYDQVGCGNSTHLP (Pro specific)DAPADFWTPQLFVDEFHAVCTALGIERYHVLGQSWGGMLGAEIAVRQPSGLVSLAICNSPASMRLWSEAAGDLRAQLPAETRAALDRHEAAGTITHPDYLQAAAEFYRRHVCRVVPTPQDFADSVAQMEAEPTVYHTMNGPNEFHVVGTLGDWSVIDRLPDVTAPVLVIAGEHDEATPKTWQPFVDHIPDVRSHVFPGTSHCTHLEKPEEFRAVVAQFLHQHDLAADARV Y. pestis Proline 48MTQQEYQNRRQALLAKMAPGSAAIIFAAPEATRSADSEYPYRQNSD iminopeptidaseFSYLTGFNEPEAVLILVKSDETHNHSVLFNRIRDLTAEIWFGRRLG (Pro Specific)QEAAPTKLAVDRALPFDEINEQLYLLLNRLDVIYHAQGQYAYADNIVFAALEKLRHGFRKNLRAPATLIDWRPWLHEMRLFKSAEEIAVLRRAGEISALAHTRAMEKCRPGMFEYQLEGEILHEFTRHGARYPAYNTIVGGGENGCILHYTENECELRDGDLVLIDAGCEYRGYAGDITRTFPVNGKFTPAQRAVYDIVLAAINKSLTLFRPGTSIREVTEEVVRIMVVGLVELGILKGDIEQLIAEQAHRPFFMHGLSHWLGMDVHDVGDYGSSDRGRILEPGMVLTVEPGLYIAPDADVPPQYRGIGIRIEDDIVITATGNENLTASVVKDPDDIEALMALNHAGENLYFQE P. furiosus 49MDTEKLMKAGEIAKKVREKAIKLARPGMLLLELAESIEKMIMELGG methionineKPAFPVNLSINEIAAHYTPYKGDTTVLKEGDYLKIDVGVHIDGFIA aminopeptidaseDTAVTVRVGMEEDELMEAAKEALNAAISVARAGVEIKELGKAIENEIRKRGFKPIVNLSGHKIERYKLHAGISIPNIYRPHDNYVLKEGDVFAIEPFATIGAGQVIEVPPTLIYMYVRDVPVRVAQARFLLAKIKREYGTLPFAYRWLQNDMPEGQLKLALKTLEKAGAIYGYPVLKEIRNGIVAQFEHTIIVEKDSVIVTQDMINKSTLE Aeromonas sobria 50HMSSPLHYVLDGIHCEPHFFTVPLDHQQPDDEETITLFGRTLCRKD ProlineRLDDELPWLLYLQGGPGFGAPRPSANGGWIKRALQEFRVLLLDQRG aminopeptidaseTGHSTPIHAELLAHLNPRQQADYLSHFRADSIVRDAELIREQLSPDHPWSLLGQSFGGFCSLTYLSLFPDSLHEVYLTGGVAPIGRSADEVYRATYQRVADKNRAFFARFPHAQAIANRLATHLQRHDVRLPNGQRLTVEQLQQQGLDLGASGAFEELYYLLEDAFIGEKLNPAFLYQVQAMQPFNTNPVFAILHELIYCEGAASHWAAERVRGEFPALAWAQGKDFAFTGEMIFPWMFEQFRELIPLKEAAHLLAEKADWGPLYDPVQLARNKVPVACAVYAEDMYVEFDYSRETLKGLSNSRAWITNEYEHNGLRVDGEQ ILDRLIRLNRDCLEPyrococcus furiosus 51 MKERLEKLVKFMDENSIDRVFIAKPVNVYYFSGTSPLGGGYIIVDGProline DEATLYVPELEYEMAKEESKLPVVKFKKFDEIYEILKNTETLGIEGAminopeptidase (X-/- TLSYSMVENFKEKSNVKEFKKIDDVIKDLRIIKTKEEIEIIEKACE Pro)IADKAVMAAIEEITEGKREREVAAKVEYLMKMNGAEKPAFDTIIASGHRSALPHGVASDKRIERGDLVVIDLGALYNHYNSDITRTIVVGSPNEKQREIYEIVLEAQKRAVEAAKPGMTAKELDSIAREIIKEYGYGDYFIHSLGHGVGLEIHEWPRISQYDETVLKEGMVITIEPGIYIPKLGGVRIEDTVLITENGAKRLTKTERELL Elizabethkingia 52MIPITTPVGNFKVWTKRFGTNPKIKVLLLHGGPAMTHEYMECFETF meningosepticaFQREGFEFYEYDQLGSYYSDQPTDEKLWNIDRFVDEVEQVRKAIHA ProlineDKENFYVLGNSWGGILAMEYALKYQQNLKGLIVANMMASAPEYVKY aminopeptidaseAEVLSKQMKPEVLAEVRAIEAKKDYANPRYTELLFPNYYAQHICRLKEWPDALNRSLKHVNSTVYTLMQGPSELGMSSDARLAKWDIKNRLHEIATPTLMIGARYDTMDPKAMEEQSKLVQKGRYLYCPNGSHLAMWD DQKVFMDGVIKFIKDVDTKSFNN. gonorrhoeae 53 MYEIKQPFHSGYLQVSEIHQIYWEESGNPDGVPVIFLHGGPGAGAS ProlinePECRGFFNPDVFRIVIIDQRGCGRSHPYACAEDNTTWDLVADIEKV IminopeptidaseREMLGIGKWLVFGGSWGSTLSLAYAQTHPERVKGLVLRGIFLCRPSETAWLNEAGGVSRIYPEQWQKFVAPIAENRRNRLIEAYHGLLFHQDEEVCLSAAKAWADWESYLIRFEPEGVDEDAYASLAIARLENHYFVNGGWLQGDKAILNNIGKIRHIPTVIVQGRYDLCTPMQSAWELSKAFPEAELRVVQAGHCAFDPPLADALVQAVEDILPRLL

TABLE 4 Non-limiting examples of non-specific aminopeptidases. SEQ NameID NO: Sequence E. coli 54MTQQPQAKYRHDYRAPDYQITDIDLTFDLDAQKTVVTAVSQAVRHG Aminopeptidase N*ASDAPLRLNGEDLKLVSVHINDEPWTAWKEEEGALVISNLPERFTL (ZincKIINEISPAANTALEGLYQSGDALCTQCEAEGFRHITYYLDRPDVL Metalloprotease)ARFTTKIIADKIKYPFLLSNGNRVAQGELENGRHWVQWQDPFPKPCYLFALVAGDFDVLRDTFTTRSGREVALELYVDRGNLDRAPWAMTSLKNSMKWDEERFGLEYDLDIYMIVAVDFFNMGAMENKGLNIFNSKYVLARTDTATDKDYLDIERVIGHEYFHNWTGNRVTCRDWFQLSLKEGLTVFRDQEFSSDLGSRAVNRINNVRTMRGLQFAEDASPMAHPIRPDMVIEMNNFYTLTVYEKGAEVIRMIHTLLGEENFQKGMQLYFERHDGSAATCDDFVQAMEDASNVDLSHFRRWYSQSGTPIVTVKDDYNPETEQYTLTISQRTPATPDQAEKQPLHIPFAIELYDNEGKVIPLQKGGHPVNSVLNVTQAEQTFVFDNVYFQPVPALLCEFSAPVKLEYKWSDQQLTFLMRHARNDFSRWDAAQSLLATYIKLNVARHQQGQPLSLPVHVADAFRAVLLDEKIDPALAAEILTLPSVNEMAELFDIIDPIAIAEVREALTRTLATELADELLAIYNANYQSEYRVEHEDIAKRTLRNACLRFLAFGETHLADVLVSKQFHEANNMTDALAALSAAVAAQLPCRDALMQEYDDKWHQNGLVMDKWFILQATSPAANVLETVRGLLQHRSFTMSNPNRIRSLIGAFAGSNPAAFHAEDGSGYLFLVEMLTDLNSRNPQVASRLIEPLIRLKRYDAKRQEKMRAALEQLKGLENLSGDLYEKITKALA P. falciparum MI 55PKIHYRKDYKPSGFIINQVTLNINIHDQETIVRSVLDMDISKHNVG aminopeptidase**EDLVFDGVGLKINEISINNKKLVEGEEYTYDNEFLTIFSKFVPKSKFAFSSEVIIHPETNYALTGLYKSKNIIVSQCEATGFRRITFFIDRPDMMAKYDVTVTADKEKYPVLLSNGDKVNEFEIPGGRHGARFNDPPLKPCYLFAVVAGDLKHLSATYITKYTKKKVELYVFSEEKYVSKLQWALECLKKSMAFDEDYFGLEYDLSRLNLVAVSDFNVGAMENKGLNIFNANSLLASKKNSIDFSYARILTVVGHEYFHQYTGNRVTLRDWFQLTLKEGLTVHRENLFSEEMTKTVTTRLSHVDLLRSVQFLEDSSPLSHPIRPESYVSMENFYTTTVYDKGSEVMRMYLTILGEEYYKKGFDIYIKKNDGNTATCEDFNYAMEQAYKMKKADNSANLNQYLLWFSQSGTPHVSFKYNYDAEKKQYSIHVNQYTKPDENQKEKKPLFIPISVGLINPENGKEMISQTTLELTKESDTFVFNNIAVKPIPSLERGESAPVYIEDQLTDEERILLLKYDSDAFVRYNSCTNIYMKQILMNYNEFLKAKNEKLESFQLTPVNAQFIDAIKYLLEDPHADAGFKSYIVSLPQDRYIINFVSNLDTDVLADTKEYIYKQIGDKLNDVYYKMFKSLEAKADDLTYFNDESHVDFDQMNMRTLRNTLLSLLSKAQYPNILNEIIEHSKSPYPSNWLTSLSVSAYFDKYFELYDKTYKLSKDDELLLQEWLKTVSRSDRKDIYEILKKLENEVLKDSKNPNDIRAVYLPFTNNLRRFHDISGKGYKLIAEVITKTDKFNPMVATQLCEPFKLWNKLDTKRQELMLNEMNTMLQEPQ ISNNLKEYLLRLTNKPuromycin-sensitive 56 MWLAAAAPSLARRLLFLGPPPPPLLLLVFSRSSRRRLHSLGLAAMPaminopeptidase EKRPFERLPADVSPINYSLCLKPDLLDFTFEGKLEAAAQVRQATNQ (“NPEPPS”)IVMNCADIDIITASYAPEGDEEIHATGFNYQNEDEKVTLSFPSTLQTGTGTLKIDFVGELNDKMKGFYRSKYTTPSGEVRYAAVTQFEATDARRAFPCWDEPAIKATFDISLVVPKDRVALSNMNVIDRKPYPDDENLVEVKFARTPVMSTYLVAFVVGEYDFVETRSKDGVCVRVYTPVGKAEQGKFALEVAAKTLPFYKDYFNVPYPLPKIDLIAIADFAAGAMENWGLVTYRETALLIDPKNSCSSSRQWVALVVGHELAHQWFGNLVTMEWWTHLWLNEGFASWIEYLCVDHCFPEYDIWTQFVSADYTRAQELDALDNSHPIEVSVGHPSEVDEIFDAISYSKGASVIRMLHDYIGDKDFKKGMNMYLTKFQQKNAATEDLWESLENASGKPIAAVMNTWTKQMGFPLIYVEAEQVEDDRLLRLSQKKFCAGGSYVGEDCPQWMVPITISTSEDPNQAKLKILMDKPEMNVVLKNVKPDQWVKLNLGTVGFYRTQYSSAMLESLLPGIRDLSLPPVDRLGLQNDLFSLARAGIISTVEVLKVMEAFVNEPNYTVWSDLSCNLGILSTLLSHTDEYEEIQEFVKDVESPIGERLGWDPKPGEGHLDALLRGLVLGKLGKAGHKATLEEARRRFKDHVEGKQILSADLRSPVYLTVLKHGDGTTLDIMLKLHKQADMQEEKNRIERVLGATLLPDLIQKVLTFALSEEVRPQDTVSVIGGVAGGSKHGRKAAWKFIKDNWEELYNRYQGGFLISRLIKLSVEGFAVDKMAGEVKAFFESHPAPSAERTIQQCCENILLNAAWLKRDAESIHQYLLQRKASPPTV NPEPPS E366V 57MWLAAAAPSLARRLLFLGPPPPPLLLLVFSRSSRRRLHSLGLAAMPEKRPFERLPADVSPINYSLCLKPDLLDFTFEGKLEAAAQVRQATNQIVMNCADIDIITASYAPEGDEEIHATGFNYQNEDEKVTLSFPSTLQTGTGTLKIDFVGELNDKMKGFYRSKYTTPSGEVRYAAVTQFEATDARRAFPCWDEPAIKATFDISLVVPKDRVALSNMNVIDRKPYPDDENLVEVKFARTPVMSTYLVAFVVGEYDFVETRSKDGVCVRVYTPVGKAEQGKFALEVAAKTLPFYKDYFNVPYPLPKIDLIAIADFAAGAMENWGLVTYRETALLIDPKNSCSSSRQWVALVVGHVLAHQWFGNLVTMEWWTHLWLNEGFASWIEYLCVDHCFPEYDIWTQFVSADYTRAQELDALDNSHPIEVSVGHPSEVDEIFDAISYSKGASVIRMLHDYIGDKDFKKGMNMYLTKFQQKNAATEDLWESLENASGKPIAAVMNTWTKQMGFPLIYVEAEQVEDDRLLRLSQKKFCAGGSYVGEDCPQWMVPITISTSEDPNQAKLKILMDKPEMNVVLKNVKPDQWVKLNLGTVGFYRTQYSSAMLESLLPGIRDLSLPPVDRLGLQNDLFSLARAGIISTVEVLKVMEAFVNEPNYTVWSDLSCNLGILSTLLSHTDFYEEIQEFVKDVESPIGERLGWDPKPGEGHLDALLRGLVLGKLGKAGHKATLEEARRRFKDHVEGKQILSADLRSPVYLTVLKHGDGTTLDIMLKLHKQADMQEEKNRIERVLGATLLPDLIQKVLTFALSEEVRPQDTVSVIGGVAGGSKHGRKAAWKFIKDNWEELYNRYQGGFLISRLIKLSVEGFAVDKMAGEVKAFFESHPAPSAERTIQQCCENILLNAAWLKRDAESIHQYLLQRKASPPTV Francisella tularensis 58MIYEFVMTDPKIKYLKDYKPSNYLIDETHLIFELDESKTRVTANLY Aminopeptidase NIVANRENRENNTLVLDGVELKLLSIKLNNKHLSPAEFAVNENQLIINNVPEKFVLQTVVEINPSANTSLEGLYKSGDVFSTQCEATGFRKITYYLDRPDVMAAFTVKIIADKKKYPIILSNGDKIDSGDISDNQHFAVWKDPFKKPCYLFALVAGDLASIKDTYITKSQRKVSLEIYAFKQDIDKCHYAMQAVKDSMKWDEDRFGLEYDLDTFMIVAVPDFNAGAMENKGLNIFNTKYIMASNKTATDKDFELVQSVVGHEYEHNWTGDRVTCRDWFQLSLKEGLTVFRDQEFTSDLNSRDVKRIDDVRIIRSAQFAEDASPMSHPIRPESYIEMNNEYTVTVYNKGAEIIRMIHTLLGEEGFQKGMKLYFERHDGQAVTCDDFVNAMADANNRDFSLFKRWYAQSGTPNIKVSENYDASSQTYSLTLEQTTLPTADQKEKQALHIPVKMGLINPEGKNIAEQVIELKEQKQTYTFENIAAKPVASLFRDFSAPVKVEHKRSEKDLLHIVKYDNNAFNRWDSLQQIATNIILNNADLNDEFLNAFKSILHDKDLDKALISNALLIPIESTIAEAMRVIMVDDIVLSRKNVVNQLADKLKDDWLAVYQQCNDNKPYSLSAEQIAKRKLKGVCLSYLMNASDQKVGTDLAQQLFDNADNMTDQQTAFTELLKSNDKQVRDNAINEFYNRWRHEDLVVNKWLLSQAQISHESALDIVKGLVNHPAYNPKNPNKVYSLIGGFGANFLQYHCKDGLGYAFMADTVLALDKFNHQVAARMARNLMSWKRYDSDRQAMMKNALEKIKASNPSKNVFEIVSKSLES Pyrococcus 59MEVRNMVDYELLKKVVEAPGVSGYEFLGIRDVVIEEIKDYVDEVKV horikoshii TETDKLGNVIAHKKGEGPKVMIAAHMDQIGLMVTHIEKNGFLRVAPIGG AminopeptidaseVDPKTLIAQRFKVWIDKGKFIYGVGASVPPHIQKPEDRKKAPDWDQIFIDIGAESKEEAEDMGVKIGTVITWDGRLERLGKHRFVSIAFDDRIAVYTILEVAKQLKDAKADVYFVATVQEEVGLRGARTSAFGIEPDYGFAIDVTIAADIPGTPEHKQVTHLGKGTAIKIMDRSVICHPTIVRWLEELAKKHEIPYQLEILLGGGTDAGAIHLTKAGVPTGALSVPARYIHSNTEVVDERDVDATVELMTKALENIHELKI T. aquaticus 60MDAFTENLNKLAELAIRVGLNLEEGQEIVATAPIEAVDFVRLLAEK Aminopeptidase TAYENGASLFTVLYGDNLIARKRLALVPEAHLDRAPAWLYEGMAKAFHEGAARLAVSGNDPKALEGLPPERVGRAQQAQSRAYRPTLSAITEFVTNWTIVPFAHPGWAKAVFPGLPEEEAVQRLWQAIFQATRVDQEDPVAAWEAHNRVLHAKVAFLNEKRFHALHFQGPGTDLTVGLAEGHLWQGGATPTKKGRLCNPNLPTEEVFTAPHRERVEGVVRASRPLALSGQLVEGLWARFEGGVAVEVGAEKGEEVLKKLLDTDEGARRLGEVALVPADNPIAKTGLVFFDTLFDENAASHIAFGQAYAENLEGRPSGEEFRRRGGNESMVHVDWMIGSEEVDVDGLLEDGTRVPLMRRGRWVI Bacillus 61MAKLDETLTMLKALTDAKGVPGNEREARDVMKTYIAPYADEVTTDG stearothermophilusLGSLIAKKEGKSGGPKVMIAGHLDEVGFMVTQIDDKGFIRFQTLGG Peptidase M28WWSQVMLAQRVTIVTKKGDITGVIGSKPPHILPSEARKKPVEIKDMFIDIGATSREEAMEWGVRPGDMIVPYFEFTVLNNEKMLLAKAWDNRIGCAVAIDVLKQLKGVDHPNTVYGVGTVQEEVGLRGARTAAQFIQPDIAFAVDVGIAGDTPGVSEKEAMGKLGAGPHIVLYDATMVSHRGLREFVIEVAEELNIPHHFDAMPGVGTDAGAIHLTGIGVPSLTIAIPTRYIHSHAAILHRDDYENTVKLLVEVIKRLDADKVKQLTFDE Vibrio cholera 62MEDKVWISMGADAVGSLNPALSESLLPHSFASGSQVWIGEVAIDEL AminopeptidaseAELSHTMHEQHNRCGGYMVHTSAQGAMAALMMPESIANFTIPAPSQQDLVNAWLPQVSADQITNTIRALSSFNNRFYTTTSGAQASDWLANEWRSLISSLPGSRIEQIKHSGYNQKSVVLTIQGSEKPDEWVIVGGHLDSTLGSHTNEQSIAPGADDDASGIASLSEIIRVLRDNNFRPKRSVALMAYAAEEVGLRGSQDLANQYKAQGKKVVSVLQLDMTNYRGSAEDIVFITDYTDSNLTQFLTTLIDEYLPELTYGYDRCGYACSDHASWHKAGFSAAMPFESKEKDYNPKIHTSQDTLANSDPTGNHAVKFTKLGLAYVIEMANAGSSQVPDDSVLQDGTAKINLSGARGTQKRFTFELSQSKPLTIQTYGGSGDVDLYVKYGSAPSKSNWDCRPYQNGNRETCSFNNAQ PGIYHVMLDGYTNYNDVALKASTQPhotobacterium 63 MEDKVWISIGSDASQTVKSVMQSNARSLLPESLASNGPVWVGQVDYhalotolerans SQLAELSHHMHEDHQRCGGYMVHSSPESAIAASNMPQSLVAFSIPEAminopeptidase ISQQDTVNAWLPQVNSQAITGTITSLTSFINRFYTTTSGAQASDWLANEWRSLSASLPNASVRQVSHFGYNQKSVVLTITGSEKPDEWIVLGGHLDSTIGSHTNEQSVAPGADDDASGIASVTEIIRVLSENNFQPKRSIAFMAYAAEEVGLRGSQDLANQYKAEGKQVISALQLDMTNYKGSVEDIVFITDYTDSNLTTFLSQLVDEYLPSLTYGFDTCGYACSDHASWHKAGESAAMPFEAKFNDYNPMIHTPNDTLQNSDPTASHAVKFTKLGLAYAIEMASTTGGTPPPTGNVLKDGVPVNGLSGATGSQVHYSFELPAQKNLQISTAGGSGDVDLYVSFGSEATKQNWDCRPYRNGNNEVCTFAGATPGTYSIMLDGYRQFSGVTLKASTQ Yersinia pestis 64MTQQPQAKYRHDYRAPDYTITDIDLDFALDAQKTTVTAVSKVKRQG AminopeptidaseNTDVTPLILNGEDLTLISVSVDGQAWPHYRQQDNTLVIEQLPADFTLTIVNDIHPATNSALEGLYLSGEALCTQCEAEGFRHITYYLDRPDVLARFTTRIVADKSRYPYLLSNGNRVGQGELDDGRHWVKWEDPFPKPSYLFALVAGDFDVLQDKFITRSGREVALEIFVDRGNLDRADWAMTSLKNSMKWDETRFGLEYDLDIYMIVAVDFFNMGAMENKGLNVFNSKYVLAKAETATDKDYLNIEAVIGHEYFHNWTGNRVTCRDWFQLSLKEGLTVFRDQEFSSDLGSRSVNRIENVRVMRAAQFAEDASPMAHAIRPDKVIEMNNFYTLTVYEKGSEVIRMMHTLLGEQQFQAGMRLYFERHDGSAATCDDFVQAMEDVSNVDLSLFRRWYSQSGTPLLTVHDDYDVEKQQYHLFVSQKTLPTADQPEKLPLHIPLDIELYDSKGNVIPLQHNGLPVHHVLNVTEAEQTFTFDNVAQKPIPSLLREFSAPVKLDYPYSDQQLTFLMQHARNEFSRWDAAQSLLATYIKLNVAKYQQQQPLSLPAHVADAFRAILLDEHLDPALAAQILTLPSENEMAELFTTIDPQAISTVHEAITRCLAQELSDELLAVYVANMTPVYRIEHGDIAKRALRNTCLNYLAFGDEEFANKLVSLQYHQADNMTDSLAALAAAVAAQLPCRDELLAAFDVRWNHDGLVMDKWFALQATSPAANVLVQVRTLLKHPAFSLSNPNRTRSLIGSFASGNPAAFHAADGSGYQFLVEILSDLNTRNPQVAARLIEPLIRLKRYDAGRQALMRKALEQLKTLDNLSGDLYEKITKALAA Vibrio anguillarum 65MEEKVWISIGGDATQTALRSGAQSLLPENLINQTSVWVGQVPVSEL AminopeptidaseATLSHEMHENHQRCGGYMVHPSAQSAMSVSAMPLNLNAFSAPEITQQTTVNAWLPSVSAQQITSTITTLTQFKNRFYTTSTGAQASNWIADHWRSLSASLPASKVEQITHSGYNQKSVMLTITGSEKPDEWVVIGGHLDSTLGSRTNESSIAPGADDDASGIAGVTEIIRLLSEQNFRPKRSIAFMAYAAEEVGLRGSQDLANRFKAEGKKVMSVMQLDMTNYQGSREDIVFITDYTDSNFTQYLTQLLDEYLPSLTYGFDTCGYACSDHASWHAVGYPAAMPFESKFNDYNPNIHSPQDTLQNSDPTGFHAVKFTKLGLAYVVEMGNASTPPTPSNQLKNGVPVNGLSASRNSKTWYQFELQEAGNLSIVLSGGSGDADLYVKYQTDADLQQYDCRPYRSGNNETCQFSNAQP GRYSILLHGYNNYSNASLVANAQSalinivibrio spYCSC6 66 MEDKKVWISIGADAQQTALSSGAQPLLAQSVAHNGQAWIGEVSESEAminopeptidase LAALSHEMHENHHRCGGYIVHSSAQSAMAASNMPLSRASFIAPAISQQALVTPWISQIDSALIVNTIDRLTDFPNRFYTTTSGAQASDWIKQRWQSLSAGLAGASVTQISHSGYNQASVMLTIEGSESPDEWVVVGGHLDSTIGSRTNEQSIAPGADDDASGIAAVTEVIRVLAQNNFQPKRSIAFVAYAAEEVGLRGSQDVANQFKQAGKDVRGVLQLDMTNYQGSAEDIVFITDYTDNQLTQYLTQLLDEYLPTLNYGFDTCGYACSDHASWHQVGYPAAMPFEAKFNDYNPNIHTPQDTLANSDSEGAHAAKFTKLGLAYTVELANADSSPNPGNELKLGEPINGLSGARGNEKYFNYRLDQSGELVIRTYGGSGDVDLYVKANGDVSTGNWDCRPYRSGNDEVCRFDNAT PGNYAVMLRGYRTYDNVSLIVEVibrio proteolyticus 67 MPPITQQATVTAWLPQVDASQITGTISSLESFTNRFYTTTSGAQASAminopeptidase I DWIASEWQALSASLPNASVKQVSHSGYNQKSVVMTITGSEAPDEWIVIGGHLDSTIGSHTNEQSVAPGADDDASGIAAVTEVIRVLSENNFQPKRSIAFMAYAAEEVGLRGSQDLANQYKSEGKNVVSALQLDMTNYKGSAQDVVFITDYTDSNFTQYLTQLMDEYLPSLTYGFDTCGYACSDHASWHNAGYPAAMPFESKFNDYNPRIHTTQDTLANSDPTGSHAKKFT QLGLAYAIEMGSATGDTPTPGNQLEVibrio proteolyticus 68 MPPITQQATVTAWLPQVDASQITGTISSLESFTNRFYTTTSGAQASAminopeptidase I DWIASEWQFLSASLPNASVKQVSHSGYNQKSVVMTITGSEAPDEWI (A55F)VIGGHLDSTIGSHTNEQSVAPGADDDASGIAAVTEVIRVLSENNFQPKRSIAFMAYAAEEVGLRGSQDLANQYKSEGKNVVSALQLDMTNYKGSAQDVVFITDYTDSNFTQYLTQLMDEYLPSLTYGFDTCGYACSDHASWHNAGYPAAMPFESKFNDYNPRIHTTQDTLANSDPTGSHAKKFT QLGLAYAIEMGSATGDTPTPGNQLEP. furiosus 69 MVDWELMKKIIESPGVSGYEHLGIRDLVVDILKDVADEVKIDKLGNAminopeptidase I VIAHFKGSAPKVMVAAHMDKIGLMVNHIDKDGYLRVVPIGGVLPETLIAQKIRFFTEKGERYGVVGVLPPHLRREAKDQGGKIDWDSIIVDVGASSREEAEEMGFRIGTIGEFAPNFTRLSEHRFATPYLDDRICLYAMIEAARQLGEHEADIYIVASVQEEIGLRGARVASFAIDPEVGIAMDVTFAKQPNDKGKIVPELGKGPVMDVGPNINPKLRQFADEVAKKYEIPLQVEPSPRPTGTDANVMQINREGVATAVLSIPIRYMHSQVELADARDVDNTIKLAKALLEELKPMDFTPLE *Cleavage efficiency (from most to least):arginine > lysine > hydrophobic residues (including alanine, leucine,methionine, and phenylalanine) > proline (see, e.g., MatthewsBiochemisty 47, 2008, 5303-5311). **Cleavage efficiency (from most toleast): leucine > alanine > arginine > phenylalanine > proline; does notcleave after glutamate and aspartate.

For the purposes of comparing two or more amino acid sequences, thepercentage of “sequence identity” between a first amino acid sequenceand a second amino acid sequence (also referred to herein as “amino acididentity”) may be calculated by dividing [the number of amino acidresidues in the first amino acid sequence that are identical to theamino acid residues at the corresponding positions in the second aminoacid sequence] by [the total number of amino acid residues in the firstamino acid sequence] and multiplying by [100], in which each deletion,insertion, substitution or addition of an amino acid residue in thesecond amino acid sequence compared to the first amino acid sequence isconsidered as a difference at a single amino acid residue (position).Alternatively, the degree of sequence identity between two amino acidsequences may be calculated using a known computer algorithm (e.g., bythe local homology algorithm of Smith and Waterman (1970) Adv. Appl.Math. 2:482c, by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. (1970) 48:443, by the search for similarity methodof Pearson and Lipman. Proc. Natl. Acad. Sci. USA (1998) 85:2444, or bycomputerized implementations of algorithms available as Blast, ClustalOmega, or other sequence alignment algorithms) and, for example, usingstandard settings. Usually, for the purpose of determining thepercentage of “sequence identity” between two amino acid sequences inaccordance with the calculation method outlined hereinabove, the aminoacid sequence with the greatest number of amino acid residues will betaken as the “first” amino acid sequence, and the other amino acidsequence will be taken as the “second” amino acid sequence.

Additionally, or alternatively, two or more sequences may be assessedfor the identity between the sequences. The terms “identical” or percent“identity” in the context of two or more nucleic acids or amino acidsequences, refer to two or more sequences or subsequences that are thesame. Two sequences are “substantially identical” if two sequences havea specified percentage of amino acid residues or nucleotides that arethe same (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.6%, 99.7%, 99.8%, or 99.9% identical) over a specified region or overthe entire sequence, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the above sequence comparison algorithms or bymanual alignment and visual inspection. Optionally, the identity existsover a region that is at least about 25, 50, 75, or 100 amino acids inlength, or over a region that is 100 to 150, 150 to 200, 100 to 200, or200 or more, amino acids in length.

Additionally, or alternatively, two or more sequences may be assessedfor the alignment between the sequences. The terms “alignment” orpercent “alignment” in the context of two or more nucleic acids or aminoacid sequences, refer to two or more sequences or subsequences that arethe same. Two sequences are “substantially aligned” if two sequenceshave a specified percentage of amino acid residues or nucleotides thatare the same (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical) over a specified regionor over the entire sequence, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the above sequence comparison algorithms or bymanual alignment and visual inspection. Optionally, the alignment existsover a region that is at least about 25, 50, 75, or 100 amino acids inlength, or over a region that is 100 to 150, 150 to 200, 100 to 200, or200 or more amino acids in length.

In addition to protein molecules, nucleic acid molecules possess avariety of advantageous properties for use as affinity reagents (e.g.,amino acid recognition molecules) in accordance with the application.

Nucleic acid aptamers are nucleic acid molecules that have beenengineered to bind desired targets with high affinity and selectivity.Accordingly, nucleic acid aptamers may be engineered to selectively binda desired type of amino acid using selection and/or enrichmenttechniques known in the art. Thus, in some embodiments, an affinityreagent comprises a nucleic acid aptamer (e.g., a DNA aptamer, an RNAaptamer). As shown in FIG. 1C, in some embodiments, a labeled affinityreagent is a labeled aptamer 104 that selectively binds one type ofterminal amino acid. For example, in some embodiments, labeled aptamer104 selectively binds one type of amino acid (e.g., a single type ofamino acid or a subset of types of amino acids) at a terminus of apolypeptide, as described herein. Although not shown, it should beappreciated that labeled aptamer 104 may be engineered to selectivelybind one type of amino acid at any position of a polypeptide (e.g., at aterminal position or at terminal and internal positions of apolypeptide) in accordance with a method of the application.

In some embodiments, a labeled affinity reagent comprises a label havingbinding-induced luminescence. For example, in some embodiments, alabeled aptamer 106 comprises a donor label 112 and an acceptor label114 and functions as illustrated in panels (I) and (II) of FIG. 1C. Asdepicted in panel (I), labeled aptamer 106 as a free molecule adopts aconformation in which donor label 112 and acceptor label 114 areseparated by a distance that limits detectable FRET between the labels(e.g., about 10 nm or more). As depicted in panel (II), labeled aptamer106 as a selectively bound molecule adopts a conformation in which donorlabel 112 and acceptor label 114 are within a distance that promotesdetectable FRET between the labels (e.g., about 10 nm or less). In yetother embodiments, labeled aptamer 106 comprises a quenching moiety andfunctions analogously to a molecular beacon, wherein luminescence oflabeled aptamer 106 is internally quenched as a free molecule andrestored as a selectively bound molecule (see, e.g., Hamaguchi, et al.(2001) Analytical Biochemistry 294, 126-131). Without wishing to bebound by theory, it is thought that these and other types of mechanismsfor binding-induced luminescence may advantageously reduce or eliminatebackground luminescence to increase overall sensitivity and accuracy ofthe methods described herein.

In addition to methods of identifying a terminal amino acid of apolypeptide, the application provides methods of sequencing polypeptidesusing labeled affinity reagents. In some embodiments, methods ofsequencing may involve subjecting a polypeptide terminus to repeatedcycles of terminal amino acid detection and terminal amino acidcleavage. For example, in some embodiments, the application provides amethod of determining an amino acid sequence of a polypeptide comprisingcontacting a polypeptide with one or more labeled affinity reagentsdescribed herein and subjecting the polypeptide to Edman degradation.

Conventional Edman degradation involves repeated cycles of modifying andcleaving the terminal amino acid of a polypeptide, wherein eachsuccessively cleaved amino acid is identified to determine an amino acidsequence of the polypeptide. As an illustrative example of aconventional Edman degradation, the N-terminal amino acid of apolypeptide is modified using phenyl isothiocyanate (PITC) to form aPITC-derivatized N-terminal amino acid. The PITC-derivatized N-terminalamino acid is then cleaved using acidic conditions, basic conditions,and/or elevated temperatures. It has also been shown that the step ofcleaving the PITC-derivatized N-terminal amino acid may be accomplishedenzymatically using a modified cysteine protease from the protozoaTrypanosoma cruzi, which involves relatively milder cleavage conditionsat a neutral or near-neutral pH. Non-limiting examples of useful enzymesare described in U.S. patent application Ser. No. 15/255,433, filed Sep.2, 2016, titled “MOLECULES AND METHODS FOR ITERATIVE POLYPEPTIDEANALYSIS AND PROCESSING.”

An example of sequencing by Edman degradation using labeled affinityreagents in accordance with the application is depicted in FIG. 1D. Insome embodiments, sequencing by Edman degradation comprises providing apolypeptide 122 that is immobilized to a surface 130 of a solid support(e.g., immobilized to a bottom or sidewall surface of a sample well)through a linker 124. In some embodiments, as described herein,polypeptide 122 is immobilized at one terminus (e.g., an amino-terminalamino acid or a carboxy-terminal amino acid) such that the otherterminus is free for detecting and cleaving of a terminal amino acid.Accordingly, in some embodiments, the reagents used in Edman degradationmethods described herein preferentially interact with terminal aminoacids at the non-immobilized (e.g., free) terminus of polypeptide 122.In this way, polypeptide 122 remains immobilized over repeated cycles ofdetecting and cleaving. To this end, in some embodiments, linker 124 maybe designed according to a desired set of conditions used for detectingand cleaving, e.g., to limit detachment of polypeptide 122 from surface130 under chemical cleavage conditions. Suitable linker compositions andtechniques for immobilizing a polypeptide to a surface are described indetail elsewhere herein.

In accordance with the application, in some embodiments, a method ofsequencing by Edman degradation comprises a step (1) of contactingpolypeptide 122 with one or more labeled affinity reagents thatselectively bind one or more types of terminal amino acids. As shown, insome embodiments, a labeled affinity reagent 108 interacts withpolypeptide 122 by selectively binding the terminal amino acid. In someembodiments, step (1) further comprises removing any of the one or morelabeled affinity reagents that do not selectively bind the terminalamino acid (e.g., the free terminal amino acid) of polypeptide 122.

In some embodiments, the method further comprises identifying theterminal amino acid of polypeptide 122 by detecting labeled affinityreagent 108. In some embodiments, detecting comprises detecting aluminescence from labeled affinity reagent 108. As described herein, insome embodiments, the luminescence is uniquely associated with labeledaffinity reagent 108, and the luminescence is thereby associated withthe type of amino acid to which labeled affinity reagent 108 selectivelybinds. As such, in some embodiments, the type of amino acid isidentified by determining one or more luminescence properties of labeledaffinity reagent 108.

In some embodiments, a method of sequencing by Edman degradationcomprises a step (2) of removing the terminal amino acid of polypeptide122. In some embodiments, step (2) comprises removing labeled affinityreagent 108 (e.g., any of the one or more labeled affinity reagents thatselectively bind the terminal amino acid) from polypeptide 122. In someembodiments, step (2) comprises modifying the terminal amino acid (e.g.,the free terminal amino acid) of polypeptide 122 by contacting theterminal amino acid with an isothiocyanate (e.g., PITC) to form anisothiocyanate-modified terminal amino acid. In some embodiments, anisothiocyanate-modified terminal amino acid is more susceptible toremoval by a cleaving reagent (e.g., a chemical or enzymatic cleavingreagent) than an unmodified terminal amino acid.

In some embodiments, step (2) comprises removing the terminal amino acidby contacting polypeptide 122 with a protease 140 that specificallybinds and cleaves the isothiocyanate-modified terminal amino acid. Insome embodiments, protease 140 comprises a modified cysteine protease.In some embodiments, protease 140 comprises a modified cysteineprotease, such as a cysteine protease from Trypanosoma cruzi (see, e.g.,Borgo, et al. (2015) Protein Science 24:571-579). In yet otherembodiments, step (2) comprises removing the terminal amino acid bysubjecting polypeptide 122 to chemical (e.g., acidic, basic) conditionssufficient to cleave the isothiocyanate-modified terminal amino acid.

In some embodiments, a method of sequencing by Edman degradationcomprises a step (3) of washing polypeptide 122 following terminal aminoacid cleavage. In some embodiments, washing comprises removing protease140. In some embodiments, washing comprises restoring polypeptide 122 toneutral pH conditions (e.g., following chemical cleavage by acidic orbasic conditions). In some embodiments, a method of sequencing by Edmandegradation comprises repeating steps (1) through (3) for a plurality ofcycles.

An example method of sequencing by Edman degradation is shown in FIG.1E. In some embodiments, a sample containing a complex mixture ofpolypeptides (e.g., a mixture of proteins) can be degraded using commonenzymes into short polypeptide fragments of approximately 6 to 40 aminoacids. In some embodiments, sequencing of this polypeptide library inaccordance with methods of the application would reveal the identity andabundance of each of the polypeptides present in the original complexmixture. As described herein and in the literature, most polypeptides inthe size range of 6 to 40 amino acids can be uniquely identified bydetermining the number and location of just four amino acids within apolypeptide chain.

Accordingly, in some embodiments, a method of sequencing by Edmandegradation may be performed using a set of labeled aptamers 150comprising four DNA aptamer types, each type recognizing a differentN-terminal amino acid. Each aptamer type may be labeled with a differentluminescent label, such that the different aptamer types can bedistinguished based on one or more luminescence properties. Forillustrative purposes, the example set of labeled aptamers 150 includes:a cysteine-specific aptamer labeled with a first luminescent label (“dye1”); a lysine-specific aptamer labeled with a second luminescent label(“dye 2”); a tryptophan-specific aptamer labeled with a thirdluminescent label (“dye 3”); and a glutamate-specific aptamer labeledwith a fourth luminescent label (“dye 4”).

In some embodiments, a method of sequencing by Edman degradation inaccordance with the application proceeds according to a process 152shown in FIG. 1E. In some embodiments, prior to step (1), singlepolypeptide molecules from a polypeptide library are immobilized to asurface of a solid support, e.g., at a bottom or sidewall surface of asample well of an array of sample wells. In some embodiments, asdescribed elsewhere herein, moieties that enable surface immobilization(e.g., biotin) or improve solubility (e.g., oligonucleotides) may bechemically or enzymatically attached to the C-terminus of thepolypeptides. To determine the sequence of each polypeptide, in someembodiments, immobilized polypeptides are subjected to repeated cyclesof N-terminal amino acid detection and N-terminal amino acid cleavage,as illustrated by process 152. In some embodiments, process 152comprises reagent addition and wash steps which are performed byinjection into a flowcell above the detection surface using an automatedfluidic system. In some embodiments, steps (1) through (4) illustrateone cycle of detection and cleavage using labeled aptamers 150.

In some embodiments, a method of sequencing by Edman degradationaccording to process 152 comprises a step (1) of flowing in a mixture offour orthogonally labeled DNA aptamers and incubating to allow theaptamers to bind to any immobilized polypeptides (e.g., polypeptidesimmobilized within a sample well of an array) that contain one of thefour correct amino acids at the N-terminus. In some embodiments, themethod further comprises washing the immobilized polypeptides to removeunbound aptamers. In some embodiments, the method further comprisesimaging the immobilized polypeptides (“Imaging step 1”). In someembodiments, the acquired images contain enough information to determinethe location of aptamer-bound polypeptides (e.g., location within anarray of sample wells) and which of the four aptamers is bound at eachlocation. In some embodiments, the method further comprises washing theimmobilized polypeptides using an appropriate buffer to remove theaptamers from the immobilized polypeptides.

In some embodiments, a method of sequencing according to process 152comprises a step (2) of flowing in a solution containing a reactivemolecule (e.g., PITC, as shown) that specifically modifies theN-terminal amine group. An isothiocyanate molecule such as PITC, in someembodiments, modifies the N-terminal amino acid into a substrate forcleavage by a modified protease such as the cysteine protease cruzainfrom Trypanosoma Cruzi.

In some embodiments, a method of sequencing according to process 152comprises a step (3) of washing the immobilized polypeptides beforeflowing in a suitable modified protease that recognizes and cleaves themodified N-terminal amino acid from the immobilized polypeptide. In someembodiments, the method comprises a step (4) of washing the immobilizedpolypeptides after enzymatic cleavage. In some embodiments, steps (1)through (4) depict one cycle of Edman degradation. Accordingly, step(1′) as shown is the start of the next reaction cycle which proceeds assteps (1′) through (4′) performed as described above for steps (1)through (4). In some embodiments, steps (1) through (4) are repeated forapproximately 20-40 cycles.

In some embodiments, a labeled isothiocyanate (e.g., a dye-labeled PITC)may be used to monitor sample loading. For example, in some embodiments,prior to subjecting a polypeptide sample to a method of sequencing asshown in process 152, the polypeptide sample is pre-conjugated with aluminescent label at a terminal end by modification of the terminal endusing a dye-labeled PITC. In this way, loading of the polypeptide sampleinto an array of sample wells may be monitored by detecting luminescencefrom the labels prior to initiating process 152. In some embodiments,the luminescence is used to determine single occupancy of sample wellsin the array (e.g., a fraction of sample wells containing a singlepolypeptide molecule), which may advantageously increase the amount ofinformation reliably obtained for a given sample. Once a desired sampleloading status is determined by luminescence, process 152 may beinitiated by chemical or enzymatic cleavage, as described, beforeproceeding with step (1).

In some embodiments, a labeled isothiocyanate (e.g., a dye-labeled PITC)may be used to monitor reaction progress for a polypeptide sample in anarray. For example, in some embodiments, step (2) comprises flowing in asolution containing a dye-labeled PITC that specifically modifies andlabels N-terminal amine groups of polypeptides in the sample. In someembodiments, luminescence from the labels may be detected during orafter step (2) to evaluate N-terminal PITC modification of polypeptidesin the sample. Accordingly, in some embodiments, luminescence is used todetermine whether or when to proceed from step (2) to step (3). In someembodiments, luminescence from the labels may be detected during orafter step (3) to evaluate N-terminal amino acid cleavage ofpolypeptides in the sample—e.g., to determine whether or when to proceedfrom step (3) to step (4).

A method of sequencing according to process 152 utilizes separatereagents for detecting and cleaving a terminal amino acid of apolypeptide. Nonetheless, in some aspects, the application provides amethod of sequencing in which a single reagent comprising a peptidasemay be used for detecting and cleaving a terminal amino acid of apolypeptide. FIG. 2 shows an example of polypeptide sequencing using aset of labeled exopeptidases 200, wherein each labeled exopeptidaseselectively binds and cleaves a different type of terminal amino acid.

As generically illustrated in the example of FIG. 2, labeledexopeptidases 200 include a lysine-specific exopeptidase comprising afirst luminescent label, a glycine-specific exopeptidase comprising asecond luminescent label, an aspartate-specific exopeptidase comprisinga third luminescent label, and a leucine-specific exopeptidasecomprising a fourth luminescent label. In accordance with certainembodiments described herein, each of labeled exopeptidases 200selectively binds and cleaves its respective amino acid only when thatamino acid is at an amino- or carboxy-terminus of a polypeptide.Accordingly, as sequencing by this approach proceeds from one terminusof a peptide toward the other, labeled exopeptidases 200 are engineeredor selected such that all reagents of the set will possess eitheraminopeptidase or carboxypeptidase activity.

As further shown in FIG. 2, process 202 schematically illustrates areal-time sequencing reaction using labeled exopeptidases 200. Panels(I) through (IX) illustrate a progression of events involving iterativedetection and cleavage at a terminal end of a polypeptide in relation toa signal output shown below, and corresponding to, the event depicted ineach panel. For illustrative purposes, a polypeptide is shown having anarbitrarily selected amino acid sequence of “KLDG . . . ” (proceedingfrom one terminus toward the other).

Panel (I) depicts the start of a sequencing reaction, wherein apolypeptide is immobilized to a surface of a solid support, such as abottom or sidewall surface of a sample well. In some embodiments,sequencing methods in accordance with the application comprise singlemolecule sequencing in real-time. In some embodiments, a plurality ofsingle molecule sequencing reactions are performed simultaneously in anarray of sample wells. In such embodiments, polypeptide immobilizationprevents diffusion of a polypeptide out of a sample well by anchoringthe polypeptide within the sample well for single molecule analysis.

Panel (II) depicts a detection event, wherein the lysine-specificexopeptidase from the set of labeled affinity reagents 200 selectivelybinds the terminal lysine residue of the polypeptide. As shown in thesignal trace below panels (I) and (II), signal output reports on thisbinding event by displaying an increase in signal intensity, which maybe used to identify the luminescent label of the lysine-specificexopeptidase to thereby identify the terminal amino acid. Panel (III)illustrates that, after selectively binding a terminal amino acid, alabeled peptidase cleaves the terminal amino acid. As a result, thesecomponents are free to diffuse away from an observation region forluminescence detection, which is reported in the signal output by a dropin signal intensity, as shown in the trace below panel (III). Panels(IV) through (IX) proceed analogously to the process as described forpanels (I) through (III). That is, a labeled exopeptidase binds andcleaves a corresponding terminal amino acid to produce a correspondingincrease and decrease, respectively, in signal output.

In some aspects, the application provides methods of polypeptidesequencing in real-time by evaluating binding interactions of terminalamino acids with labeled amino acid recognition molecules (e.g., labeledaffinity reagents) and a labeled cleaving reagent (e.g., a labelednon-specific exopeptidase). FIG. 3A shows an example of a method ofsequencing in which discrete binding events give rise to signal pulsesof a signal output 300. The inset panel of FIG. 3A illustrates a generalscheme of real-time sequencing by this approach. As shown, a labeledaffinity reagent 310 selectively associates with (e.g., binds to) anddissociates from a terminal amino acid (shown here as lysine), whichgives rise to a series of pulses in signal output 300 which may be usedto identify the terminal amino acid. In some embodiments, the series ofpulses provide a pulsing pattern (e.g., a characteristic pattern) whichmay be diagnostic of the identity of the corresponding terminal aminoacid.

Without wishing to be bound by theory, labeled affinity reagent 310selectively binds according to a binding affinity (K_(D)) defined by anassociation rate, or an “on” rate, of binding (k_(on)) and adissociation rate, or an “off” rate, of binding (k_(off)). The rateconstants k_(off) and k_(on) are the critical determinants of pulseduration (e.g., the time corresponding to a detectable binding event)and interpulse duration (e.g., the time between detectable bindingevents), respectively. In some embodiments, these rates can beengineered to achieve pulse durations and pulse rates (e.g., thefrequency of signal pulses) that give the best sequencing accuracy.

As shown in the inset panel, a sequencing reaction mixture furthercomprises a labeled non-specific exopeptidase 320 comprising aluminescent label that is different than that of labeled affinityreagent 310. In some embodiments, labeled non-specific exopeptidase 320is present in the mixture at a concentration that is less than that oflabeled affinity reagent 310. In some embodiments, labeled non-specificexopeptidase 320 displays broad specificity such that it cleaves most orall types of terminal amino acids. Accordingly, a dynamic sequencingapproach can involve monitoring affinity reagent binding at a terminusof a polypeptide over the course of a degradation reaction catalyzed byexopeptidase cleavage activity.

As illustrated by the progress of signal output 300, in someembodiments, terminal amino acid cleavage by labeled non-specificexopeptidase 320 gives rise to a signal pulse, and these events occurwith lower frequency than the binding pulses of a labeled affinityreagent 310. In this way, amino acids of a polypeptide may be countedand/or identified in a real-time sequencing process. As furtherillustrated in signal output 300, in some embodiments, a plurality oflabeled affinity reagents may be used, each with a diagnostic pulsingpattern (e.g., characteristic pattern) which may be used to identify acorresponding terminal amino acid. For example, in some embodiments,different characteristic patterns (as illustrated by each of lysine,phenylalanine, and glutamine in signal output 300) correspond to theassociation of more than one labeled affinity reagent with differenttypes of terminal amino acids. As described herein, it should beappreciated that a single affinity reagent that associates with morethan one type of amino acid may be used in accordance with theapplication. Accordingly, in some embodiments, different characteristicpatterns correspond to the association of one labeled affinity reagentwith different types of terminal amino acids.

As described herein, signal pulse information may be used to identify anamino acid based on a characteristic pattern in a series of signalpulses. In some embodiments, a characteristic pattern comprises aplurality of signal pulses, each signal pulse comprising a pulseduration. In some embodiments, the plurality of signal pulses may becharacterized by a summary statistic (e.g., mean, median, time decayconstant) of the distribution of pulse durations in a characteristicpattern. In some embodiments, the mean pulse duration of acharacteristic pattern is between about 1 millisecond and about 10seconds (e.g., between about 1 ms and about 1 s, between about 1 ms andabout 100 ms, between about 1 ms and about 10 ms, between about 10 msand about 10 s, between about 100 ms and about 10 s, between about 1 sand about 10 s, between about 10 ms and about 100 ms, or between about100 ms and about 500 ms). In some embodiments, different characteristicpatterns corresponding to different types of amino acids in a singlepolypeptide may be distinguished from one another based on astatistically significant difference in the summary statistic. Forexample, in some embodiments, one characteristic pattern may bedistinguishable from another characteristic pattern based on adifference in mean pulse duration of at least 10 milliseconds (e.g.,between about 10 ms and about 10 s, between about 10 ms and about 1 s,between about 10 ms and about 100 ms, between about 100 ms and about 10s, between about 1 s and about 10 s, or between about 100 ms and about 1s). It should be appreciated that, in some embodiments, smallerdifferences in mean pulse duration between different characteristicpatterns may require a greater number of pulse durations within eachcharacteristic pattern to distinguish one from another with statisticalconfidence.

As detailed above, a real-time sequencing process as illustrated by FIG.3A can generally involve cycles of terminal amino acid recognition andterminal amino acid cleavage, where the relative occurrence ofrecognition and cleavage can be controlled by a concentrationdifferential between a labeled affinity reagent 310 and a labelednon-specific exopeptidase 320. In some embodiments, the concentrationdifferential can be optimized such that the number of signal pulsesdetected during recognition of an individual amino acid provides adesired confidence interval for identification. For example, if aninitial sequencing reaction provides signal data with too few signalpulses between cleavage events to permit determination of characteristicpatterns with a desired confidence interval, the sequencing reaction canbe repeated using a decreased concentration of non-specific exopeptidaserelative to affinity reagent.

In some embodiments, polypeptide sequencing in accordance with theapplication may be carried out by contacting a polypeptide with asequencing reaction mixture comprising one or more amino acidrecognition molecules (e.g., affinity reagents) and/or one or morecleaving reagents (e.g., exopeptidases). In some embodiments, asequencing reaction mixture comprises an amino acid recognition moleculeat a concentration of between about 10 nM and about 10 μM. In someembodiments, a sequencing reaction mixture comprises a cleaving reagentat a concentration of between about 500 nM and about 500 μM.

In some embodiments, a sequencing reaction mixture comprises an aminoacid recognition molecule at a concentration of between about 100 nM andabout 10 μM, between about 250 nM and about 10 μM, between about 100 nMand about 1 μM, between about 250 nM and about 1 μM, between about 250nM and about 750 nM, or between about 500 nM and about 1 μM. In someembodiments, a sequencing reaction mixture comprises an amino acidrecognition molecule at a concentration of about 100 nM, about 250 nM,about 500 nM, about 750 nM, or about 1 μM.

In some embodiments, a sequencing reaction mixture comprises a cleavingreagent at a concentration of between about 500 nM and about 250 μM,between about 500 nM and about 100 μM, between about 1 μM and about 100μM, between about 500 nM and about 50 μM, between about 1 μM and about100 μM, between about 10 μM and about 200 μM, or between about 10 μM andabout 100 μM. In some embodiments, a sequencing reaction mixturecomprises a cleaving reagent at a concentration of about 1 μM, about 5μM, about 10 μM, about 30 μM, about 50 μM, about 70 μM, or about 100 μM.

In some embodiments, a sequencing reaction mixture comprises an aminoacid recognition molecule at a concentration of between about 10 nM andabout 10 μM, and a cleaving reagent at a concentration of between about500 nM and about 500 μM. In some embodiments, a sequencing reactionmixture comprises an amino acid recognition molecule at a concentrationof between about 100 nM and about 1 μM, and a cleaving reagent at aconcentration of between about 1 μM and about 100 μM. In someembodiments, a sequencing reaction mixture comprises an amino acidrecognition molecule at a concentration of between about 250 nM andabout 1 μM, and a cleaving reagent at a concentration of between about10 μM and about 100 μM. In some embodiments, a sequencing reactionmixture comprises an amino acid recognition molecule at a concentrationof about 500 nM, and a cleaving reagent at a concentration of betweenabout 25 μM and about 75 μM.

In some embodiments, a sequencing reaction mixture comprises an aminoacid recognition molecule and a cleaving reagent in a ratio of about500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 75:1,about 50:1, about 25:1, about 10:1, about 5:1, about 2:1, or about 1:1.In some embodiments, a sequencing reaction mixture comprises an aminoacid recognition molecule and a cleaving reagent in a ratio of betweenabout 10:1 and about 200:1. In some embodiments, a sequencing reactionmixture comprises an amino acid recognition molecule and a cleavingreagent in a ratio of between about 50:1 and about 150:1.

While the example illustrated by FIG. 3A relates to a sequencing processusing a labeled cleaving reagent, the sequencing process is not intendedto be limited in this respect. As described elsewhere herein, theinventors have demonstrated single-molecule sequencing using anunlabeled cleaving reagent. In some embodiments, the approximatefrequency with which a cleaving reagent removes successive terminalamino acids is known, e.g., based on a known activity and/orconcentration of the enzyme being used. In some embodiments, terminalamino acid cleavage by the reagent is inferred, e.g., based on signaldetected for amino acid recognition or a lack of signal detected. Theinventors have recognized further techniques for controlling real-timesequencing reactions, which may be used in combination with, oralternatively to, the concentration differential approach as described.

An example of a temperature-dependent real-time sequencing process isshown in FIG. 3B. Panels (I) through (III) illustrate a sequencingreaction involving cycles of temperature-dependent terminal amino acidrecognition and terminal amino acid cleavage. Each cycle of thesequencing reaction is carried out over two temperature ranges: a firsttemperature range (“T₁”) that is optimal for affinity reagent activityover exopeptidase activity (e.g., to promote terminal amino acidrecognition), and a second temperature range (“T₂”) that is optimal forexopeptidase activity over affinity reagent activity (e.g., to promoteterminal amino acid cleavage). The sequencing reaction progresses byalternating the reaction mixture temperature between the firsttemperature range T₁ (to initiate amino acid recognition) and the secondtemperature range T₂ (to initiate amino acid cleavage). Accordingly,progression of a temperature-dependent sequencing process iscontrollable by temperature, and alternating between differenttemperature ranges (e.g., between T₁ and T₂) may be carried throughmanual or automated processes. In some embodiments, affinity reagentactivity (e.g., binding affinity (K_(D)) for an amino acid) within thefirst temperature range T₁ as compared to the second temperature rangeT₂ is increased by at least 10-fold, at least 100-fold, at least1,000-fold, at least 10,000-fold, at least 100,000-fold, or more. Insome embodiments, exopeptidase activity (e.g., rate of substrateconversion to cleavage product) within the second temperature range T₂as compared to the first temperature range T₁ is increased by at least2-fold, 10-fold, at least 25-fold, at least 50-fold, at least 100-fold,at least 1,000-fold, or more.

In some embodiments, the first temperature range T₁ is lower than thesecond temperature range T₂. In some embodiments, the first temperaturerange T₁ is between about 15° C. and about 40° C. (e.g., between about25° C. and about 35° C., between about 15° C. and about 30° C., betweenabout 20° C. and about 30° C.). In some embodiments, the secondtemperature range T₂ is between about 40° C. and about 100° C. (e.g.,between about 50° C. and about 90° C., between about 60° C. and about90° C., between about 70° C. and about 90° C.). In some embodiments, thefirst temperature range T₁ is between about 20° C. and about 40° C.(e.g., approximately 30° C.), and the second temperature range T₂ isbetween about 60° C. and about 100° C. (e.g., approximately 80° C.).

In some embodiments, the first temperature range T₁ is higher than thesecond temperature range T₂. In some embodiments, the first temperaturerange T₁ is between about 40° C. and about 100° C. (e.g., between about50° C. and about 90° C., between about 60° C. and about 90° C., betweenabout 70° C. and about 90° C.). In some embodiments, the secondtemperature range T₂ is between about 15° C. and about 40° C. (e.g.,between about 25° C. and about 35° C., between about 15° C. and about30° C., between about 20° C. and about 30° C.). In some embodiments, thefirst temperature range T₁ is between about 60° C. and about 100° C.(e.g., approximately 80° C.), and the second temperature range T₂ isbetween about 20° C. and about 40° C. (e.g., approximately 30° C.).

Panel (I) depicts a sequencing reaction mixture at a temperature that iswithin a first temperature range T₁ which is optimal for affinityreagent activity over exopeptidase activity. For illustrative purposes,a polypeptide of amino acid sequence “KFVAG . . . ” is shown. When thereaction mixture temperature is within the first temperature range T₁,labeled affinity reagents in the mixture are activated (e.g., renatured)to initiate amino acid recognition by associating with the polypeptideterminus. Also within the first temperature range T₁, labeledexopeptidases in the mixture are inactivated (e.g., denatured) toprevent amino acid cleavage during recognition. In panel (I), a firstaffinity reagent is shown reversibly associating with lysine at thepolypeptide terminus, while a labeled exopeptidase (e.g., Pfuaminopeptidase I (Pfu API)) is shown denatured. In some embodiments,amino acid recognition occurs for a predetermined duration of timebefore initiating cleavage of the amino acid. In some embodiments, aminoacid recognition occurs for a duration of time required to reach adesired confidence interval for identification before initiatingcleavage of the amino acid. Following amino acid recognition, thereaction proceeds by changing the temperature of the mixture to within asecond temperature range T₂.

Panel (II) depicts the sequencing reaction mixture at a temperature thatis within a second temperature range T₂ which is optimal forexopeptidase activity over affinity reagent activity. For illustrativepurposes of this example, the second temperature range T₂ is higher thanthe first temperature range T₁, although it should be appreciated thatreagent activity may be optimized for any desired temperature range.Accordingly, progression from panel (I) to panel (II) is carried out byraising the reaction mixture temperature using a suitable source ofheat. When the reaction mixture reaches a temperature that is within thesecond temperature range T₂, labeled exopeptidases in the mixture areactivated (e.g., renatured) to initiate terminal amino acid cleavage byexopeptidase activity. Also within the second temperature range Tz,labeled affinity reagents in the mixture are inactivated (e.g.,denatured) to prevent amino acid recognition during cleavage. In panel(II), a labeled exopeptidase is shown cleaving the terminal lysineresidue, while labeled affinity reagents are denatured. In someembodiments, amino acid cleavage occurs for a predetermined duration oftime before initiating recognition of a successive amino acid at thepolypeptide terminus. In some embodiments, amino acid cleavage occursfor a duration of time required to detect cleavage before initiatingrecognition of a successive amino acid. Following amino acid cleavage,the reaction proceeds by changing the temperature of the mixture towithin the first temperature range T₁.

Panel (III) depicts the beginning of the next cycle in the sequencingreaction, wherein the reaction mixture temperature has been reduced backto within the first temperature range T₁. Accordingly, in this example,progression from panel (II) to panel (III) can be carried out byremoving the reaction mixture from the source of heat or otherwisecooling the reaction mixture (e.g., actively or passively) to within thefirst temperature range T₁. As shown, labeled affinity reagents arerenatured, including a second affinity reagent that reversiblyassociates with phenylalanine at the polypeptide terminus, while thelabeled exopeptidase is shown denatured. The sequencing reactioncontinues by further cycling between amino acid recognition and aminoacid cleavage in a temperature-dependent fashion as illustrated by thisexample.

Accordingly, a dynamic sequencing approach can involve reaction cyclingthat is controlled at the level of protein activity or function of oneor more proteins within a reaction mixture. It should be appreciatedthat the temperature-dependent polypeptide sequencing process depictedin FIG. 3B and described above may be illustrative of a general approachto polypeptide sequencing by controllable cycling of condition-dependentrecognition and cleavage. For example, in some embodiments, theapplication provides a luminescence-dependent sequencing process usingluminescence-activated reagents. In some embodiments, aluminescence-dependent sequencing process involves cycles ofluminescence-dependent amino acid recognition and cleavage. Each cycleof the sequencing reaction may be carried out by exposing a sequencingreaction mixture to two different luminescent conditions: a firstluminescent condition that is optimal for affinity reagent activity overexopeptidase activity (e.g., to promote amino acid recognition), and asecond luminescent condition that is optimal for exopeptidase activityover affinity reagent activity (e.g., to promote amino acid cleavage).The sequencing reaction progresses by alternating between exposing thereaction mixture to the first luminescent condition (to initiate aminoacid recognition) and exposing the reaction mixture to the secondluminescent condition (to initiate amino acid cleavage). By way ofexample and not limitation, in some embodiments, the two differentluminescent conditions comprise a first wavelength and a secondwavelength.

In some aspects, the application provides methods of polypeptidesequencing in real-time by evaluating binding interactions of one ormore labeled affinity reagents with terminal and internal amino acidsand binding interactions of a labeled non-specific exopeptidase withterminal amino acids. FIG. 4 shows an example of a method of sequencingin which the method described and illustrated for the approach in FIGS.3A-3B is modified by using a labeled affinity reagent 410 thatselectively binds to and dissociates from one type of amino acid (shownhere as lysine) at both terminal and internal positions (FIG. 4, insetpanel). As described in the previous approach, the selective bindinggives rise to a series of pulses in signal output 400. In this approach,however, the series of pulses occur at a rate that is determined by thenumber of the type of amino acid throughout the polypeptide.Accordingly, in some embodiments, the rate of pulsing corresponding tobinding events would be diagnostic of the number of cognate amino acidscurrently present in the polypeptide.

As in the previous approach, a labeled non-specific peptidase 420 wouldbe present at a relatively lower concentration than labeled affinityreagent 410, e.g., to give optimal time windows in between cleavageevents (FIG. 4, inset panel). Additionally, in certain embodiments,uniquely identifiable luminescent label of labeled non-specificpeptidase 420 would indicate when cleavage events have occurred. As thepolypeptide undergoes iterative cleavage, the rate of pulsingcorresponding to binding by labeled affinity reagent 410 would drop in astep-wise manner whenever a terminal amino acid is cleaved by labelednon-specific peptidase 420. This concept is illustrated by plot 402,which generally depicts pulse rate as a function of time, with cleavageevents in time denoted by arrows. Thus, in some embodiments, amino acidsmay be identified—and polypeptides thereby sequenced—in this approachbased on a pulsing pattern and/or on the rate of pulsing that occurswithin a pattern detected between cleavage events.

In some embodiments, terminal polypeptide sequence information (e.g.,determined as described herein) can be combined with polypeptidesequence information obtained from one or more other sources. Forexample, terminal polypeptide sequence information could be combinedwith internal polypeptide sequence information. In some embodiments,internal polypeptide sequence information can be obtained using one ormore amino acid recognition molecules that associate with internal aminoacids, as described herein. Internal or other polypeptide sequenceinformation can be obtained before or during a polypeptide degradationprocess. In some embodiments, sequence information obtained from thesemethods can be combined with polypeptide sequence information usingother techniques, e.g., sequence information obtained using one or moreinternal amino acid recognition molecules.

Shielded Recognition Molecules

In accordance with embodiments described herein, single-moleculepolypeptide sequencing methods can be carried out by illuminating asurface-immobilized polypeptide with excitation light, and detectingluminescence produced by a label attached to an amino acid recognitionmolecule (e.g., a labeled affinity reagent). In some cases, radiativeand/or non-radiative decay produced by the label can result inphotodamage to the polypeptide. For example, FIG. 5A illustrates anexample sequencing reaction in which a recognition molecule is shownassociated with a polypeptide immobilized to a surface.

In the presence of excitation illumination, the label can producefluorescence through radiative decay which results in a detectableassociation event. However, in some cases, the label producesnon-radiative decay which can result in the formation of reactive oxygenspecies 500. The reactive oxygen species 500 can eventually damage theimmobilized peptide, such that the reaction ends before obtainingcomplete sequence information for the polypeptide. This photodamage canoccur, for example, at the exposed polypeptide terminus (top openarrow), at an internal position (middle open arrow), or at the surfacelinker attaching the polypeptide to the surface (bottom open arrow).

The inventors have found that photodamage can be mitigated andrecognition times extended by incorporation of a shielding element intoan amino acid recognition molecule. FIG. 5B illustrates an examplesequencing reaction using a shielded recognition molecule that includesa shield 502. Shield 502 forms a covalent or non-covalent linkage groupthat provides increased distance between the label and polypeptide, suchthat damaging effects from reactive oxygen species 500 can be reduceddue to free radical decay over the label-polypeptide separationdistance. Shield 502 can also provide a steric barrier that shields thepolypeptide from the label by absorbing damage from reactive oxygenspecies 500 and radiative and/or non-radiative decay.

Without wishing to be bound by theory, it is thought that a shield,positioned between a recognition component and a label component, canabsorb, deflect, or otherwise block radiative and/or non-radiative decayemitted by the label component. In some embodiments, the shield preventsor limits the extent to which one or more labels (e.g., luminescentlabels) interact with one or more amino acid recognition molecules. Insome embodiments, the shield prevents or limits the extent to which oneor more labels interact with one or more molecules associated with anamino acid recognition molecule (e.g., a polypeptide associated with therecognition molecule). Accordingly, in some embodiments, the term shieldcan generally refer to a protective or shielding effect that is providedby some portion of a linkage group formed between a recognitioncomponent and a label component.

In some embodiments, a shield is attached to one or more amino acidrecognition molecules (e.g., a recognition component) and to one or morelabels (e.g., a label component). In some embodiments, the recognitionand label components are attached at non-adjacent sites on the shield.For example, one or more amino acid recognition molecules can beattached to a first side of the shield, and one or more labels can beattached to a second side of the shield, where the first and secondsides of the shield are distant from each other. In some embodiments,the attachment sites are on approximately opposite sides of the shield.

The distance between the site at which a shield is attached to arecognition molecule and the site at which the shield is attached to alabel can be a linear measurement through space or a non-linearmeasurement across the surface of the shield. The distance between therecognition molecule and label attachment sites on a shield can bemeasured by modeling the three-dimensional structure of the shield. Insome embodiments, this distance can be at least 2 nm, at least 4 nm, atleast 6 nm, at least 8 nm, at least 10 nm, at least 12 nm, at least 15nm, at least 20 nm, at least 30 nm, at least 40 nm, or more.Alternatively, the relative positions of the recognition molecule andlabel on a shield can be described by treating the structure of theshield as a quadratic surface (e.g., ellipsoid, elliptic cylinder). Insome embodiments, the recognition molecule and label attachment sitesare separated by a distance that is at least one eighth of the distancearound an ellipsoidal shape representing the shield. In someembodiments, the recognition molecule and label are separated by adistance that is at least one quarter of the distance around anellipsoidal shape representing the shield. In some embodiments, therecognition molecule and label are separated by a distance that is atleast one third of the distance around an ellipsoidal shape representingthe shield. In some embodiments, the recognition molecule and label areseparated by a distance that is one half of the distance around anellipsoidal shape representing the shield.

The size of a shield should be such that a label is unable or unlikelyto directly contact the polypeptide when the amino acid recognitionmolecule is associated with the polypeptide. The size of a shield shouldalso be such that an attached label is detectable when the amino acidrecognition molecule is associated with the polypeptide. For example,the size should be such that an attached luminescent label is within anillumination volume to be excited.

It should be appreciated that there are a variety of parameters by whicha practitioner could evaluate shielding effects. Generally, the effectsof a shielding element can be evaluated by conducting a comparativeassessment between a composition having the shielding element and acomposition lacking the shielding element. For example, a shieldingelement can increase recognition time of an amino acid recognitionmolecule. In some embodiments, recognition time refers to the length oftime in which association events between the recognition molecule and apolypeptide are observable in a polypeptide sequencing reaction asdescribed herein. In some embodiments, recognition time is increased byabout 10-25%, 25-50%, 50-75%, 75-100%, or more than 100%, for example byabout 2-fold, 3-fold, 4-fold, 5-fold, or more, relative to a polypeptidesequencing reaction performed under the same conditions, with theexception that the amino acid recognition molecule lacks the shieldingelement but is otherwise similar or identical. In some embodiments, ashielding element can increase sequencing accuracy and/or sequence readlength (e.g., by at least 5%, at least 10%, at least 15%, at least 25%or more, relative to a sequencing reaction performed under comparativeconditions as described above).

Accordingly, in some aspects, the application provides shieldedrecognition molecules comprising at least one amino acid recognitionmolecule, at least one detectable label, and a shielding element (e.g.,a “shield”) that forms a covalent or non-covalent linkage group betweenthe recognition molecule and label. In some embodiments, a shieldingelement is at least 2 nm, at least 5 nm, at least 10 nm, at least 12 nm,at least 15 nm, at least 20 nm, or more, in length (e.g., in an aqueoussolution). In some embodiments, a shielding element is between about 2nm and about 100 nm in length (e.g., between about 2 nm and about 50 nm,between about 10 nm and about 50 nm, between about 20 nm and about 100nm).

In some embodiments, a shield (e.g., shielding element) forms a covalentor non-covalent linkage group between one or more amino acid recognitionmolecules (e.g., a recognition component) and one or more labels (e.g.,a label component). As used herein, in some embodiments, covalent andnon-covalent linkages or linkage groups refer to the nature of theattachments of the recognition and label components to the shield.

In some embodiments, a covalent linkage, or a covalent linkage group,refers to a shield that is attached to each of the recognition and labelcomponents through a covalent bond or a series of contiguous covalentbonds. Covalent attachment one or both components can be achieved bycovalent conjugation methods known in the art. For example, in someembodiments, click chemistry techniques (e.g., copper-catalyzed,strain-promoted, copper-free click chemistry, etc.) can be used toattach one or both components to the shield. Such methods generallyinvolve conjugating one reactive moiety to another reactive moiety toform one or more covalent bonds between the reactive moieties.Accordingly, in some embodiments, a first reactive moiety of a shieldcan be contacted with a second reactive moiety of a recognition or labelcomponent to form a covalent attachment. Examples of reactive moietiesinclude, without limitation, reactive amines, azides, alkynes, nitrones,alkenes (e.g., cycloalkenes), tetrazines, tetrazoles, and other reactivemoieties suitable for click reactions and similar coupling techniques.

In some embodiments, a non-covalent linkage, or a non-covalent linkagegroup, refers to a shield that is attached to one or both of therecognition and label components through one or more non-covalentcoupling means, including but not limited to receptor-ligandinteractions and oligonucleotide strand hybridization. Examples ofreceptor-ligand interactions are provided herein and include, withoutlimitation, protein-protein complexes, protein-ligand complexes,protein-aptamer complexes, and aptamer-nucleic acid complexes. Variousconfigurations and strategies for oligonucleotide strand hybridizationare described herein and are known in the art (see, e.g., U.S. PatentPublication No. 2019/0024168).

In some embodiments, shield 502 comprises a polymer, such as abiomolecule or a dendritic polymer. FIG. 5C depicts examples of polymershields and configurations of shielded recognition molecules of theapplication. A first shielded construct 504 shows an example of aprotein shield 530. In some embodiments, protein shield 530 forms acovalent linkage group between a recognition molecule and a label. Forexample, in some embodiments, protein shield 530 is attached to each ofthe recognition molecule and label through one or more covalent bonds,e.g., by covalent attachment through a side-chain of a natural orunnatural amino acid of protein shield 530. In some embodiments, proteinshield 530 forms a non-covalent linkage group between a recognitionmolecule and a label. For example, in some embodiments, protein shield530 is a monomeric or multimeric protein comprising one or moreligand-binding sites. In some embodiments, a non-covalent linkage groupis formed through one or more ligand moieties bound to the one or moreligand-binding sites. Additional examples of non-covalent linkagesformed by protein shields are described elsewhere herein.

A second shielded construct 506 shows an example of a double-strandednucleic acid shield comprising a first oligonucleotide strand 532hybridized with a second oligonucleotide strand 534. As shown, in someembodiments, the double-stranded nucleic acid shield can comprise arecognition molecule attached to first oligonucleotide strand 532, and alabel attached to second oligonucleotide strand 534. In this way, thedouble-stranded nucleic acid shield forms a non-covalent linkage groupbetween the recognition molecule and the label through oligonucleotidestrand hybridization. In some embodiments, a recognition molecule and alabel can be attached to the same oligonucleotide strand, which canprovide a single-stranded nucleic acid shield or a double-strandednucleic acid shield through hybridization with another oligonucleotidestrand. In some embodiments, strand hybridization can provide increasedrigidity within a linkage group to further enhance separation betweenthe recognition molecule and the label.

Where shielding element 502 comprises a nucleic acid, the separationdistance between a label and a recognition molecule can be measured bythe distance between attachment sites on the nucleic acid (e.g., directattachment or indirect attachment, such as through one or moreadditional shield polymers). In some embodiments, the distance betweenattachment sites on a nucleic acid can be measured by the number ofnucleotides within the nucleic acid that occur between the label and therecognition molecule. It should be understood that the number ofnucleotides can refer to either the number of nucleotide bases in asingle-stranded nucleic acid or the number of nucleotide base pairs in adouble-stranded nucleic acid.

Accordingly, in some embodiments, the attachment site of a recognitionmolecule and the attachment site of a label can be separated by between5 and 200 nucleotides (e.g., between 5 and 150 nucleotides, between 5and 100 nucleotides, between 5 and 50 nucleotides, between 10 and 100nucleotides). It should be appreciated that any position in a nucleicacid can serve as an attachment site for a recognition molecule, alabel, or one or more additional polymer shields. In some embodiments,an attachment site can be at or approximately at the 5′ or 3′ end, or atan internal position along a strand of the nucleic acid.

The non-limiting configuration of second shielded construct 506illustrates an example of a shield that forms a non-covalent linkagethrough strand hybridization. A further example of non-covalent linkageis illustrated by a third shielded construct 508 comprising anoligonucleotide shield 536. In some embodiments, oligonucleotide shield536 is a nucleic acid aptamer that binds a recognition molecule to forma non-covalent linkage. In some embodiments, the recognition molecule isa nucleic acid aptamer, and oligonucleotide shield 536 comprises anoligonucleotide strand that hybridizes with the aptamer to form anon-covalent linkage.

A fourth shielded construct 510 shows an example of a dendritic polymershield 538. As used herein, in some embodiments, a dendritic polymerrefers generally to a polyol or a dendrimer. Polyols and dendrimers havebeen described in the art, and may include branched dendritic structuresoptimized for a particular configuration. In some embodiments, dendriticpolymer shield 538 comprises polyethylene glycol, tetraethylene glycol,poly(amidoamine), poly(propyleneimine), poly(propyleneamine),carbosilane, poly(L-lysine), or a combination of one or more thereof.

A dendrimer, or dendron, is a repetitively branched molecule that istypically symmetric around the core and that may adopt a sphericalthree-dimensional morphology. See, e.g., Astruc et al. (2010) Chem. Rev.110:1857. Incorporation of such structures into a shield of theapplication can provide for a protective effect through the stericinhibition of contacts between a label and one or more biomoleculesassociated therewith (e.g., a recognition molecule and/or a polypeptideassociated with the recognition molecule). Refinement of the chemicaland physical properties of the dendrimer through variation in primarystructure of the molecule, including potential functionalization of thedendrimer surface, allows the shielding effects to be adjusted asdesired. Dendrimers may be synthesized by a variety of techniques usinga wide range of materials and branching reactions, as is known in theart. Such synthetic variation allows the properties of the dendrimer tobe customized as necessary. Examples of polyol and dendrimer compoundswhich can be used in accordance with shields of the application include,without limitation, compounds described in U.S. Patent Publication No.20180346507.

FIG. 5D depicts further example configurations of shielded recognitionmolecules of the application. A protein-nucleic acid construct 512 showsan example of a shield comprising more than one polymer in the form of aprotein and a double-stranded nucleic acid. In some embodiments, theprotein portion of the shield is attached to the nucleic acid portion ofthe shield through a covalent linkage. In some embodiments, theattachment is through a non-covalent linkage. For example, in someembodiments, the protein portion of the shield is a monovalent ormultivalent protein that forms at least one non-covalent linkage througha ligand moiety attached to a ligand-binding site of the monovalent ormultivalent protein. In some embodiments, the protein portion of theshield comprises an avidin protein.

In some embodiments, a shielded recognition molecule of the applicationis an avidin-nucleic acid construct 514. In some embodiments,avidin-nucleic acid construct 514 includes a shield comprising an avidinprotein 540 and a double-stranded nucleic acid. As described herein,avidin protein 540 may be used to form a non-covalent linkage betweenone or more amino acid recognition molecules and one or more labels,either directly or indirectly, such as through one or more additionalshield polymers described herein.

Avidin proteins are biotin-binding proteins, generally having a biotinbinding site at each of four subunits of the avidin protein. Avidinproteins include, for example, avidin, streptavidin, traptavidin,tamavidin, bradavidin, xenavidin, and homologs and variants thereof. Insome cases, the monomeric, dimeric, or tetrameric form of the avidinprotein can be used. In some embodiments, the avidin protein of anavidin protein complex is streptavidin in a tetrameric form (e.g., ahomotetramer). In some embodiments, the biotin binding sites of anavidin protein provide attachment sites for one or more amino acidrecognition molecules, one or more labels, and/or one or more additionalshield polymers described herein.

An illustrative diagram of an avidin protein complex is shown in theinset panel of FIG. 5D. As shown in the inset panel, avidin protein 540can include a binding site 542 at each of four subunits of the proteinwhich can be bound to a biotin moiety (shown as white circles). Themultivalency of avidin protein 540 can allow for various linkageconfigurations, which are generally shown for illustrative purposes. Forexample, in some embodiments, a biotin linkage moiety 544 can be used toprovide a single point of attachment to avidin protein 540. In someembodiments, a bis-biotin linkage moiety 546 can be used to provide twopoints of attachment to avidin protein 540. As illustrated byavidin-nucleic acid construct 514, an avidin protein complex may beformed by two bis-biotin linkage moieties, which form atrans-configuration to provide an increased separation distance betweena recognition molecule and a label.

Various further examples of avidin protein shield configurations areshown. A first avidin construct 516 shows an example of an avidin shieldattached to a recognition molecule through a bis-biotin linkage moietyand to two labels through separate biotin linkage moieties. A secondavidin construct 518 shows an example of an avidin shield attached totwo recognition molecules through separate biotin linkage moieties andto a label through a bis-biotin linkage moiety. A third avidin construct520 shows an example of an avidin shield attached to two recognitionmolecules through separate biotin linkage moieties and to a labelednucleic acid through a biotin linkage moiety of each strand of thenucleic acid. A fourth avidin construct 522 shows an example of anavidin shield attached to a recognition molecule and to a labelednucleic acid through separate bis-biotin linkage moieties. As shown, thelabel is further shielded from the recognition molecule by a dendriticpolymer between the label and nucleic acid.

It should be appreciated that the example configurations of shieldedrecognition molecules shown in FIGS. 5A-5D are provided for illustrativepurposes. The inventors have conceived of various other shieldconfigurations using one or more different polymers that form a covalentor non-covalent linkage between recognition and label components of ashielded recognition molecule. By way of example, FIG. 5E illustratesthe modularity of shield configuration in accordance with theapplication.

As shown at the top of FIG. 5E, a shielded recognition moleculegenerally comprises a recognition component 550, a shielding element552, and a label component 554. For ease of illustration, recognitioncomponent 550 is depicted as one amino acid recognition molecule, andlabel component 554 is depicted as one label. It should be appreciatedthat shielded recognition molecules of the application can compriseshielding element 552 attached to one or more amino acid recognitionmolecules and one or more labels. Where recognition component 550comprises more than one recognition molecule, each recognition moleculecan be attached to shielding element 552 at one or more attachment siteson shielding element 552. Where label component 554 comprises more thanone label, each label can be attached to shielding element 552 at one ormore attachment sites on shielding element 552.

In some embodiments, shielding element 552 comprises a protein 560. Insome embodiments, protein 560 is a monovalent or multivalent protein. Insome embodiments, protein 560 is a monomeric or multimeric protein, suchas a protein homodimer, protein heterodimer, protein oligomer, or otherproteinaceous molecule. In some embodiments, shielding element 552comprises a protein complex formed by a protein non-covalently bound toat least one other molecule. For example, in some embodiments, shieldingelement 552 comprises a protein-protein complex 562. In someembodiments, protein-protein complex 562 comprises one proteinaceousmolecule specifically bound to another proteinaceous molecule. In someembodiments, protein-protein complex 562 comprises an antibody orantibody fragment (e.g., scFv) bound to an antigen. In some embodiments,protein-protein complex 562 comprises a receptor bound to a proteinligand. Additional examples of protein-protein complexes include,without limitation, trypsin-aprotinin, barnase-barstar, and colicinE9-Im9 immunity protein.

In some embodiments, shielding element 552 comprises a protein-ligandcomplex 564. In some embodiments, protein-ligand complex 564 comprises amonovalent protein and a non-proteinaceous ligand moiety. For example,in some embodiments, protein-ligand complex 564 comprises an enzymebound to a small-molecule inhibitor moiety. In some embodiments,protein-ligand complex 564 comprises a receptor bound to anon-proteinaceous ligand moiety.

In some embodiments, shielding element 552 comprises a multivalentprotein complex formed by a multivalent protein non-covalently bound toone or more ligand moieties. In some embodiments, shielding element 552comprises an avidin protein complex formed by an avidin proteinnon-covalently bound to one or more biotin linkage moieties. Constructs566, 568, 570, and 572 provide illustrative examples of avidin proteincomplexes, any one or more of which may be incorporated into shieldingelement 552.

In some embodiments, shielding element 552 comprises a two-way avidincomplex 566 comprising an avidin protein bound to two bis-biotin linkagemoieties. In some embodiments, shielding element 552 comprises athree-way avidin complex 568 comprising an avidin protein bound to twobiotin linkage moieties and a bis-biotin linkage moiety. In someembodiments, shielding element 552 comprises a four-way avidin complex570 comprising an avidin protein bound to four biotin linkage moieties.

In some embodiments, shielding element 552 comprises an avidin proteincomprising one or two non-functional binding sites engineered into theavidin protein. For example, in some embodiments, shielding element 552comprises a divalent avidin complex 572 comprising an avidin proteinbound to a biotin linkage moiety at each of two subunits, where theavidin protein comprises a non-functional ligand-binding site 548 ateach of two other subunits. As shown, in some embodiments, divalentavidin complex 572 comprises a trans-divalent avidin protein, although acis-divalent avidin protein may be used depending on a desiredimplementation. In some embodiments, the avidin protein is a trivalentavidin protein. In some embodiments, the trivalent avidin proteincomprises non-functional ligand-binding site 548 at one subunit and isbound to three biotin linkage moieties, or one biotin linkage moiety andone bis-biotin linkage moiety, at the other subunits.

In some embodiments, shielding element 552 comprises a dendritic polymer574. In some embodiments, dendritic polymer 574 is a polyol or adendrimer, as described elsewhere herein. In some embodiments, dendriticpolymer 574 is a branched polyol or a branched dendrimer. In someembodiments, dendritic polymer 574 comprises a monosaccharide-TEG, adisaccharide, an N-acetyl monosaccharide, a TEMPO-TEG, a trolox-TEG, ora glycerol dendrimer. Examples of polyols useful in accordance withshielded recognition molecules of the application include polyetherpolyols and polyester polyols, e.g., polyethylene glycol, polypropyleneglycol, and similar such polymers well known in the art. In someembodiments, dendritic polymer 574 comprises a compound of the followingformula: —(CH₂CH₂O)_(n)—, where n is an integer from 1 to 500,inclusive. In some embodiments, dendritic polymer 574 comprises acompound of the following formula: —(CH₂CH₂O)_(n)—, wherein n is aninteger from 1 to 100, inclusive.

In some embodiments, shielding element 552 comprises a nucleic acid. Insome embodiments, the nucleic acid is single-stranded. In someembodiments, label component 554 is attached directly or indirectly toone end of the single-stranded nucleic acid (e.g., the 5′ end or the 3′end) and recognition component 550 is attached directly or indirectly tothe other end of the single-stranded nucleic acid (e.g., the 3′ end orthe 5′ end). For example, the single-stranded nucleic acid can comprisea label attached to the 5′ end of the nucleic acid and an amino acidrecognition molecule attached to the 3′ end of the nucleic acid.

In some embodiments, shielding element 552 comprises a double-strandednucleic acid 576. As shown, in some embodiments, double-stranded nucleicacid 576 can form a non-covalent linkage between recognition component550 and label component 554 through strand hybridization. However, insome embodiments, double-stranded nucleic acid 576 can form a covalentlinkage between recognition component 550 and label component 554through attachment to the same oligonucleotide strand. In someembodiments, label component 554 is attached directly or indirectly toone end of the double-stranded nucleic acid and recognition component550 is attached directly or indirectly to the other end of thedouble-stranded nucleic acid. For example, the double-stranded nucleicacid can comprise a label attached to the 5′ end of one strand and anamino acid recognition molecule attached to the 5′ end of the otherstrand.

In some embodiments, shielding element 552 comprises a nucleic acid thatforms one or more structural motifs which can be useful for increasingsteric bulk of the shield. Examples of nucleic acid structural motifsinclude, without limitation, stem-loops, three-way junctions (e.g.,formed by two or more stem-loop motifs), four-way junctions (e.g.,Holliday junctions), and bulge loops.

In some embodiments, shielding element 552 comprises a nucleic acid thatforms a stem-loop 578. A stem-loop, or hairpin loop, is an unpaired loopof nucleotides on an oligonucleotide strand that is formed when theoligonucleotide strand folds and forms base pairs with another sectionof the same strand. In some embodiments, the unpaired loop of stem-loop578 comprises three to ten nucleotides. Accordingly, stem-loop 578 canbe formed by two regions of an oligonucleotide strand having invertedcomplementary sequences that hybridize to form a stem, where the tworegions are separated by the three to ten nucleotides that form theunpaired loop. In some embodiments, the stem of stem-loop 578 can bedesigned to have one or more G/C nucleotides, which can provide addedstability with the addition hydrogen bonding interaction that formscompared to A/T nucleotides. In some embodiments, the stem of stem-loop578 comprises G/C nucleotides immediately adjacent to an unpaired loopsequence. In some embodiments, the stem of stem-loop 578 comprises G/Cnucleotides within the first 2, 3, 4, or 5 nucleotides adjacent to anunpaired loop sequence. In some embodiments, an unpaired loop ofstem-loop 578 comprises one or more attachment sites. In someembodiments, an attachment site occurs at an abasic site in the unpairedloop. In some embodiments, an attachment site occurs at a base of theunpaired loop.

In some embodiments, stem-loop 578 is formed by a double-strandednucleic acid. As described herein, in some embodiments, thedouble-stranded nucleic acid can form a non-covalent linkage groupthrough strand hybridization of first and second oligonucleotidestrands. However, in some embodiments, shielding element 552 comprises asingle-stranded nucleic acid that forms a stem-loop motif, e.g., toprovide a covalent linkage group. In some embodiments, shielding element552 comprises a nucleic acid that forms two or more stem-loop motifs.For example, in some embodiments, the nucleic acid comprises twostem-loop motifs. In some embodiments, a stem of one stem-loop motif isadjacent to the stem of the other such that the motifs together form athree-way junction. In some embodiments, shielding element 552 comprisesa nucleic acid that forms a four-way junction 578. In some embodiments,four-way junction 578 is formed through hybridization of two or moreoligonucleotide strands (e.g., 2, 3, or 4 oligonucleotide strands).

In some embodiments, shielding element 552 comprises one or morepolymers selected from 560, 562, 564, 566, 568, 570, 572, 574, 576, 578,and 580 of FIG. 5E. It should be appreciated that the linkage moietiesand attachment sites shown on each of 560, 562, 564, 566, 568, 570, 572,574, 576, 578, and 580 are shown for illustrative purposes and are notintended to depict a preferred linkage or attachment site configuration.

In some aspects, the application provides an amino acid recognitionmolecule of Formula (I):

A-(Y)_(n)-D  (I),

wherein: A is an amino acid binding component comprising at least oneamino acid recognition molecule; each instance of Y is a polymer thatforms a covalent or non-covalent linkage group; n is an integer from 1to 10, inclusive; and D is a label component comprising at least onedetectable label. In some embodiments, the application provides acomposition comprising a soluble amino acid recognition molecule ofFormula (I).

In some embodiments, A comprises a plurality of amino acid recognitionmolecules. In some embodiments, each amino acid recognition molecule ofthe plurality is attached to a different attachment site on Y. In someembodiments, at least two amino acid recognition molecules of theplurality are attached to a single attachment site on Y. In someembodiments, the amino acid recognition molecule is a recognitionprotein or a nucleic acid aptamer, e.g., as described elsewhere herein.

In some embodiments, the detectable label is a luminescent label or aconductivity label. In some embodiments, the luminescent label comprisesat least one fluorophore dye molecule. In some embodiments, D comprises20 or fewer fluorophore dye molecules. In some embodiments, the ratio ofthe number of fluorophore dye molecules to the number of amino acidrecognition molecules is between 1:1 and 20:1. In some embodiments, theluminescent label comprises at least one FRET pair comprising a donorlabel and an acceptor label. In some embodiments, the ratio of the donorlabel to the acceptor label is 1:1, 2:1, 3:1, 4:1, or 5:1. In someembodiments, the ratio of the acceptor label to the donor label is 1:1,2:1, 3:1, 4:1, or 5:1.

In some embodiments, D is less than 200 Å in diameter. In someembodiments, —(Y)_(n)— is at least 2 nm in length. In some embodiments,—(Y), is at least 5 nm in length. In some embodiments, —(Y)_(n)— is atleast 10 nm in length. In some embodiments, each instance of Y isindependently a biomolecule, a polyol, or a dendrimer. In someembodiments, the biomolecule is a nucleic acid, a polypeptide, or apolysaccharide.

In some embodiments, the amino acid recognition molecule is of one ofthe following formulae:

A-Y¹-(Y)_(m)-D or A-(Y)_(m)—Y¹-D,

wherein: Y¹ is a nucleic acid or a polypeptide; and m is an integer from0 to 10, inclusive.

In some embodiments, the nucleic acid comprises a first oligonucleotidestrand. In some embodiments, the nucleic acid comprises a secondoligonucleotide strand hybridized with the first oligonucleotide strand.In some embodiments, the nucleic acid forms a covalent linkage throughthe first oligonucleotide strand. In some embodiments, the nucleic acidforms a non-covalent linkage through the hybridized first and secondoligonucleotide strands.

In some embodiments, the polypeptide is a monovalent or multivalentprotein. In some embodiments, the monovalent or multivalent proteinforms at least one non-covalent linkage through a ligand moiety attachedto a ligand-binding site of the monovalent or multivalent protein. Insome embodiments, A, Y, or D comprises the ligand moiety.

In some embodiments, the amino acid recognition molecule is of one ofthe following formulae:

A-(Y)_(m)Y²-D or A-Y²-(Y)_(m)-D,

wherein: Y² is a polyol or dendrimer; and m is an integer from 0 to 10,inclusive. In some embodiments, the polyol or dendrimer comprisespolyethylene glycol, tetraethylene glycol, poly(amidoamine),poly(propyleneimine), poly(propyleneamine), carbosilane, poly(L-lysine),or a combination of one or more thereof.

In some aspects, the application provides an amino acid recognitionmolecule of Formula (II):

A-Y¹D  (II),

wherein: A is an amino acid binding component comprising at least oneamino acid recognition molecule; Y¹ is a nucleic acid or a polypeptide;D is a label component comprising at least one detectable label. In someembodiments, when Y¹ is a nucleic acid, the nucleic acid forms acovalent or non-covalent linkage group. In some embodiments, when Y¹ isa polypeptide, the polypeptide forms a non-covalent linkage groupcharacterized by a dissociation constant (K_(D)) of less than 50×10⁻⁹M.

In some embodiments, Y¹ is a nucleic acid comprising a firstoligonucleotide strand. In some embodiments, the nucleic acid comprisesa second oligonucleotide strand hybridized with the firstoligonucleotide strand. In some embodiments, A is attached to the firstoligonucleotide strand, and wherein D is attached to the secondoligonucleotide strand. In some embodiments, A is attached to a firstattachment site on the first oligonucleotide strand, and wherein D isattached to a second attachment site on the first oligonucleotidestrand. In some embodiments, each oligonucleotide strand of the nucleicacid comprises fewer than 150, fewer than 100, or fewer than 50nucleotides.

In some embodiments, Y¹ is a monovalent or multivalent protein. In someembodiments, the monovalent or multivalent protein forms at least onenon-covalent linkage through a ligand moiety attached to aligand-binding site of the monovalent or multivalent protein. In someembodiments, at least one of A and D comprises the ligand moiety. Insome embodiments, the polypeptide is an avidin protein (e.g., avidin,streptavidin, traptavidin, tamavidin, bradavidin, xenavidin, or ahomolog or variant thereof). In some embodiments, the ligand moiety is abiotin moiety.

In some embodiments, the amino acid recognition molecule is of one ofthe following formulae:

A-Y¹-(Y)_(n)-D or A-(Y)_(n)—Y¹-D,

wherein: each instance of Y is a polymer that forms a covalent ornon-covalent linkage group; and n is an integer from 1 to 10, inclusive.In some embodiments, each instance of Y is independently a biomolecule,a polyol, or a dendrimer.

In other aspects, the application provides an amino acid recognitionmolecule comprising: a nucleic acid; at least one amino acid recognitionmolecule attached to a first attachment site on the nucleic acid; and atleast one detectable label attached to a second attachment site on thenucleic acid. In some embodiments, the nucleic acid forms a covalent ornon-covalent linkage group between the at least one amino acidrecognition molecule and the at least one detectable label.

In some embodiments, the nucleic acid is a double-stranded nucleic acidcomprising a first oligonucleotide strand hybridized with a secondoligonucleotide strand. In some embodiments, the first attachment siteis on the first oligonucleotide strand, and wherein the secondattachment site is on the second oligonucleotide strand. In someembodiments, the at least one amino acid recognition molecule isattached to the first attachment site through a protein that forms acovalent or non-covalent linkage group between the at least one aminoacid recognition molecule and the nucleic acid. In some embodiments, theat least one detectable label is attached to the second attachment sitethrough a protein that forms a covalent or non-covalent linkage groupbetween the at least one detectable label and the nucleic acid. In someembodiments, the first and second attachment sites are separated bybetween 5 and 100 nucleotide bases or nucleotide base pairs on thenucleic acid.

In yet other aspects, the application provides an amino acid recognitionmolecule comprising: a multivalent protein comprising at least twoligand-binding sites; at least one amino acid recognition moleculeattached to the protein through a first ligand moiety bound to a firstligand-binding site on the protein; and at least one detectable labelattached to the protein through a second ligand moiety bound to a secondligand-binding site on the protein.

In some embodiments, the multivalent protein is an avidin proteincomprising four ligand-binding sites. In some embodiments, theligand-binding sites are biotin binding sites, and wherein the ligandmoieties are biotin moieties. In some embodiments, at least one of thebiotin moieties is a bis-biotin moiety, and wherein the bis-biotinmoiety is bound to two biotin binding sites on the avidin protein. Insome embodiments, the at least one amino acid recognition molecule isattached to the protein through a nucleic acid comprising the firstligand moiety. In some embodiments, the at least one detectable label isattached to the protein through a nucleic acid comprising the secondligand moiety.

As described elsewhere herein, shielded recognition molecules of theapplication may be used in a polypeptide sequencing method in accordancewith the application, or any method known in the art. For example, insome embodiments, a shielded recognition molecule provided herein may beused in an Edman-type degradation reaction provided herein, orconventionally known in the art, which can involve iterative cycling ofmultiple reaction mixtures in a polypeptide sequencing reaction. In someembodiments, a shielded recognition molecule provided herein may be usedin a dynamic sequencing reaction of the application, which involvesamino acid recognition and degradation in a single reaction mixture.

Sequencing by Degradation of Labeled Polypeptides

In some aspects, the application provides a method of sequencing apolypeptide by identifying a unique combination of amino acidscorresponding to a known polypeptide sequence. For example, FIG. 6 showsa method of sequencing by detecting selectively labeled amino acids of alabeled polypeptide 600. In some embodiments, labeled polypeptide 600comprises selectively modified amino acids such that different aminoacid types comprise different luminescent labels. As used herein, unlessotherwise indicated, a labeled polypeptide refers to a polypeptidecomprising one or more selectively labeled amino acid sidechains.Methods of selective labeling and details relating to the preparationand analysis of labeled polypeptides are known in the art (see, e.g.,Swaminathan, et al. PLoS Comput Biol. 2015, 11(2):e1004080).

As shown, in some embodiments, labeled polypeptide 600 is immobilizedand exposed to an excitation source. An aggregate luminescence fromlabeled polypeptide 600 is detected and, in some embodiments, exposureto luminescence over time results in a loss in detected signal due toluminescent label degradation (e.g., degradation due to photobleaching).In some embodiments, labeled polypeptide 600 comprises a uniquecombination of selectively labeled amino acids that give rise to aninitial detected signal. As generically illustrated, degradation ofluminescent labels over time results in a corresponding decrease in adetected signal for the photobleached labeled polypeptide 602. In someembodiments, the signal can be deconvoluted by analysis of one or moreluminescence properties (e.g., signal deconvolution by luminescencelifetime analysis). In some embodiments, the unique combination ofselectively labeled amino acids of labeled polypeptide 600 have beencomputationally precomputed and empirically verified—e.g., based onknown polypeptide sequences of a proteome. In some embodiments, thecombination of detected amino acid labels are compared against adatabase of known sequences of a proteome of an organism to identify aparticular polypeptide of the database corresponding to labeledpolypeptide 600.

In some embodiments, the approach illustrated in FIG. 6 may be modifiedby determining an optimal sample concentration for performing asequencing reaction that maximizes sampling in massively parallelanalysis. In some embodiments, the concentration is selected so that adesired fraction of the sample wells of an array (e.g., 30%) areoccupied at any given time. Without wishing to be bound by theory, it isthought that while a polypeptide is bleached over a period of time, thesame well continues to be available for further analysis. Throughdiffusion, approximately 30% of the sample wells of an array can be usedfor analysis every 3 minutes. As an illustrative example, in a millionsample well chip, 6,000,000 polypeptides per hour may be sampled, or24,000,000 over a 4 hour period.

In some aspects, the application provides a method of sequencing apolypeptide by detecting luminescence of a labeled polypeptide which issubjected to repeated cycles of terminal amino acid modification andcleavage. For example, FIG. 7 shows a method of sequencing a labeledpolypeptide by Edman degradation in accordance with the application. Insome embodiments, the method generally proceeds as described herein forother methods of sequencing by Edman degradation. For example, in someembodiments, steps (1) and (2) shown in FIG. 7 may be performed asdescribed elsewhere herein for terminal amino acid modification andterminal amino acid cleavage, respectively, in an Edman degradationreaction.

As shown in the example depicted in FIG. 7, in some embodiments, themethod comprises a step of (1) modifying the terminal amino acid of alabeled polypeptide. As described elsewhere herein, in some embodiments,modifying comprises contacting the terminal amino acid with anisothiocyanate (e.g., PITC) to form an isothiocyanate-modified terminalamino acid. In some embodiments, an isothiocyanate modification 710converts the terminal amino acid to a form that is more susceptible toremoval by a cleaving reagent (e.g., a chemical or enzymatic cleavingreagent, as described herein). Accordingly, in some embodiments, themethod comprises a step of (2) removing the modified terminal amino acidusing chemical or enzymatic means detailed elsewhere herein for Edmandegradation.

In some embodiments, the method comprises repeating steps (1) through(2) for a plurality of cycles, during which luminescence of the labeledpolypeptide is detected, and cleavage events corresponding to theremoval of a labeled amino acid from the terminus may be detected as adecrease in detected signal. In some embodiments, no change in signalfollowing step (2) as shown in FIG. 7 identifies an amino acid ofunknown type. Accordingly, in some embodiments, partial sequenceinformation may be determined by evaluating a signal detected followingstep (2) during each sequential round by assigning an amino acid type bya determined identity based on a change in detected signal oridentifying an amino acid type as unknown based on no change in adetected signal.

In some aspects, a method of sequencing a polypeptide in accordance withthe application comprises sequencing by processive enzymatic cleavage ofa labeled polypeptide, as generally illustrated in FIGS. 8A-8C. Asshown, in some embodiments, a labeled polypeptide is subjected todegradation using a modified processive exopeptidase that continuouslycleaves a terminal amino acid from one terminus to another terminus.Exopeptidases are described in detail elsewhere herein. FIG. 8A depictsan example in which a labeled polypeptide 800 is subjected todegradation by an immobilized processive exopeptidase 810. FIG. 8Bdepicts an example in which an immobilized labeled polypeptide 820 issubjected to degradation by a processive exopeptidase 830.

FIG. 8C schematically illustrates an example of a real-time sequencingprocess performed in accordance with the method depicted in FIG. 8B. Asshown, panels (I) through (IV) show a progression of labeled polypeptidedegradation, with a corresponding signal trace over time shown beloweach panel. As shown, each cleavage event corresponding to a labeledamino acid gives rise to a concomitant drop in signal. In someembodiments, the rate of processivity of processive exopeptidase 830 isknown, such that the timing between a detected decrease in signal may beused to calculate the number of unlabeled amino acids between eachdetection event. For example, if a polypeptide of 40 amino acids wascleaved in such a way that an amino acid was removed every second, alabeled polypeptide having 3 signals would show all 3 initially (panel(I)), then 2 (panel (II)), then 1 (panel (III)), and finally no signal.In this way, the order of the labeled amino acids can be determined.Accordingly, these methods may be used to determine partial sequenceinformation, e.g., for proteomic analysis based on polypeptide fragmentsequencing.

In some embodiments, single molecule protein sequencing can be achievedusing an ATP-based Forster resonance energy transfer (FRET) scheme(e.g., with one or more labeled cofactors), for example as illustratedin FIG. 9. In some embodiments, sequencing by cofactor-based FRET can beperformed using an immobilized ATP-dependent protease, donor-labeledATP, and acceptor-labeled amino acids of a polypeptide substrate. Insome embodiments, amino acids can be labeled with acceptors, and the oneor more cofactors can be labeled with donors.

For example, in some embodiments, extracted proteins are denatured, andcysteines and lysines are labeled with fluorescent dyes. In someembodiments, an engineered version of a protein translocase (e.g.,bacterial ClpX) is used to bind to individual substrate proteins, unfoldthem, and translocate them through its nano-channel. In someembodiments, the translocase is labeled with a donor dye, and FREToccurs between the donor on the translocase and two or more distinctacceptor dyes on a substrate when the substrate passes through thenano-channel. The order of the labeled amino acids can then bedetermined from the FRET signal. In some embodiments, one or more of thefollowing non-limiting labeled ATP analogues shown in Table 5 can beused.

TABLE 5 Non-limiting examples of labeled ATP analogues.Phosphate-labeled ATP:

Ribose-labeled ATP:

Base-labeled ATP:

Preparation of Samples for Sequencing

A polypeptide sample can be modified prior to sequencing. In someembodiments, the N-terminal amino acid or the C-terminal amino acid of apolypeptide is modified. FIG. 10A illustrates a non-limiting example ofterminal end modification for preparing terminally modified polypeptidesfrom a protein sample. In step (1), protein sample 1000 is fragmented toproduce polypeptide fragments 1002. A polypeptide can be fragmented bycleaving (e.g., chemically) and/or digesting (e.g., enzymatically, forexample using a peptidase, for example trypsin) a polypeptide ofinterest. Fragmentation can be performed before or after labeling. Insome embodiments, fragmentation is performed after labeling of wholeproteins. One or more amino acids can be labeled before or aftercleavage to produce labeled polypeptides. In some embodiments,polypeptides are size selected after chemical or enzymaticfragmentation. In some embodiments, smaller polypeptides (e.g., <2 kDa)are removed and larger polypeptides are retained for sequence analysis.Size selection can be achieved using a technique such as gel filtration,SEC, dialysis, PAGE gel extraction, microfluidic tension flow, or anyother suitable technique. In step (2), the N-termini or C-termini ofpolypeptide fragments 1002 are modified to produce terminally modifiedpolypeptides 1004. In some embodiments, modification comprises adding animmobilization moiety. In some embodiments, modification comprisesadding a coupling moiety.

Accordingly, provided herein are methods of modifying terminal ends ofproteins and polypeptides with moieties that enable immobilization to asurface (e.g., a surface of a sample well on a chip used for proteinanalysis). In some embodiments, such methods comprise modifying aterminal end of a labeled polypeptide to be analyzed in accordance withthe application. In yet other embodiments, such methods comprisemodifying a terminal end of a protein or enzyme that degrades ortranslocates a protein or polypeptide substrate in accordance with theapplication.

In some embodiments, a carboxy-terminus of a protein or polypeptide ismodified in a method comprising: (i) blocking free carboxylate groups ofthe protein or polypeptide; (ii) denaturing the protein or polypeptide(e.g., by heat and/or chemical means); (iii) blocking free thiol groupsof the protein or polypeptide; (iv) digesting the protein or polypeptideto produce at least one polypeptide fragment comprising a freeC-terminal carboxylate group; and (v) conjugating (e.g., chemically) afunctional moiety to the free C-terminal carboxylate group. In someembodiments, the method further comprises, after (i) and before (ii),dialyzing a sample comprising the protein or polypeptide.

In some embodiments, a carboxy-terminus of a protein or polypeptide ismodified in a method comprising: (i) denaturing the protein orpolypeptide (e.g., by heat and/or chemical means); (ii) blocking freethiol groups of the protein or polypeptide; (iii) digesting the proteinor polypeptide to produce at least one polypeptide fragment comprising afree C-terminal carboxylate group; (iv) blocking the free C-terminalcarboxylate group to produce at least one polypeptide fragmentcomprising a blocked C-terminal carboxylate group; and (v) conjugating(e.g., enzymatically) a functional moiety to the blocked C-terminalcarboxylate group. In some embodiments, the method further comprises,after (iv) and before (v), dialyzing a sample comprising the protein orpolypeptide.

In some embodiments, blocking free carboxylate groups refers to achemical modification of these groups which alters chemical reactivityrelative to an unmodified carboxylate. Suitable carboxylate blockingmethods are known in the art and should modify side-chain carboxylategroups to be chemically different from a carboxy-terminal carboxylategroup of a polypeptide to be functionalized. In some embodiments,blocking free carboxylate groups comprises esterification or amidationof free carboxylate groups of a polypeptide. In some embodiments,blocking free carboxylate groups comprises methyl esterification of freecarboxylate groups of a polypeptide, e.g., by reacting the polypeptidewith methanolic HCl. Additional examples of reagents and techniquesuseful for blocking free carboxylate groups include, without limitation,4-sulfo-2,3,5,6-tetrafluorophenol (STP) and/or a carbodiimide such asN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC),uronium reagents, diazomethane, alcohols and acid for Fischeresterification, the use of N-hydroxylsuccinimide (NHS) to form NHSesters (potentially as an intermediate to subsequent ester or amineformation), or reaction with carbonyldiimidazole (CDI) or the formationof mixed anhydrides, or any other method of modifying or blockingcarboxylic acids, potentially through the formation of either esters oramides.

In some embodiments, blocking free thiol groups refers to a chemicalmodification of these groups which alters chemical reactivity relativeto an unmodified thiol. In some embodiments, blocking free thiol groupscomprises reducing and alkylating free thiol groups of a protein orpolypeptide. In some embodiments, reduction and alkylation is carriedout by contacting a polypeptide with dithiothreitol (DTT) and one orboth of iodoacetamide and iodoacetic acid. Examples of additional andalternative cysteine-reducing reagents which may be used are well knownand include, without limitation, 2-mercaptoethanol, Tris(2-carboxyehtyl) phosphine hydrochloride (TCEP), tributylphosphine,dithiobutylamine (DTBA), or any reagent capable of reducing a thiolgroup. Examples of additional and alternative cysteine-blocking (e.g.,cysteine-alkylating) reagents which may be used are well known andinclude, without limitation, acrylamide, 4-vinylpyridine,N-Ethylmalemide (NEM), N-ε-maleimidocaproic acid (EMCA), or any reagentthat modifies cysteines so as to prevent disulfide bond formation.

In some embodiments, digestion comprises enzymatic digestion. In someembodiments, digestion is carried out by contacting a protein orpolypeptide with an endopeptidase (e.g., trypsin) under digestionconditions. In some embodiments, digestion comprises chemical digestion.Examples of suitable reagents for chemical and enzymatic digestion areknown in the art and include, without limitation, trypsin, chemotrypsin,Lys-C, Arg-C, Asp-N, Lys-N, BNPS-Skatole, CNBr, caspase, formic acid,glutamyl endopeptidase, hydroxylamine, iodosobenzoic acid, neutrophilelastase, pepsin, proline-endopeptidase, proteinase K, staphylococcalpeptidase I, thermolysin, and thrombin.

In some embodiments, the functional moiety comprises a biotin molecule.In some embodiments, the functional moiety comprises a reactive chemicalmoiety, such as an alkynyl. In some embodiments, conjugating afunctional moiety comprises biotinylation of carboxy-terminalcarboxy-methyl ester groups by carboxypeptidase Y, as known in the art.

In some embodiments, a solubilizing moiety is added to a polypeptide.FIG. 10B illustrates a non-limiting example of a solubilizing moietyadded to a terminal amino acid of a polypeptide, for example using aprocess of conjugating a solubilizing linker to the polypeptide.

In some embodiments, a terminally modified polypeptide 1010 comprising alinker conjugating moiety 1012 is conjugated to a solubilizing linker1020 comprising a polypeptide conjugating moiety 1022. In someembodiments, the solubilizing linker comprises a solubilizing polymer,such as a biomolecule (e.g., shown as stippled shape). In someembodiments, a resulting linker-conjugated polypeptide 1030 comprising alinkage 1032 formed between 1012 and 1022 further comprises a surfaceconjugating moiety 1034. Accordingly, in some embodiments methods andcompositions provided herein are useful for modifying terminal ends ofpolypeptides with moieties that increase their solubility. In someembodiments, a solubilizing moiety is useful for small polypeptides thatresult from fragmentation (e.g., enzymatic fragmentation, for exampleusing trypsin) and that are relatively insoluble. For example, in someembodiments, short polypeptides in a polypeptide pool can be solubilizedby conjugating a polymer (e.g., a short oligo, a sugar, or other chargedpolymer) to the polypeptides.

In some embodiments, one or more surfaces of a sample well (e.g.,sidewalls of a sample well) can be modified. A non-limiting example ofpassivation and/or antifouling of a sample well sidewall is shown inFIG. 10C where an example schematic of a sample well is illustrated withmodified surfaces which may be used to promote single moleculeimmobilization to a bottom surface. In some embodiments, 1040 is SiO₂.In some embodiments, 1042 is a polypeptide conjugating moiety (e.g.,TCO, tetrazine, N₃, alkyne, aldehyde, NCO, NHS, thiol, alkene, DBCO,BCN, TPP, biotin, or other suitable conjugating moiety). In someembodiments, 1050 is TiO₂ or Al₂O₃. In some embodiments, 1052 is ahydrophobic C₄₋₁₈ molecule, a polytetrafluoroethylene compound (e.g.,(CF₂)₄₋₁₂), a polyol, such as a polyethylene glycol (e.g., PEG₃₋₁₀₀),polypropylene glycol, polyoxyethylene glycol, or combinations orvariations thereof, or a zwitterion, such as sulfobetaine. In someembodiments, 1060 is Si. In some embodiments, 1070 is Al. In someembodiments, 1080 is TiN.

Luminescent Labels

As used herein, a luminescent label is a molecule that absorbs one ormore photons and may subsequently emit one or more photons after one ormore time durations. In some embodiments, the term is usedinterchangeably with “label” or “luminescent molecule” depending oncontext. A luminescent label in accordance with certain embodimentsdescribed herein may refer to a luminescent label of a labeled affinityreagent, a luminescent label of a labeled peptidase (e.g., a labeledexopeptidase, a labeled non-specific exopeptidase), a luminescent labelof a labeled peptide, a luminescent label of a labeled cofactor, oranother labeled composition described herein. In some embodiments, aluminescent label in accordance with the application refers to a labeledamino acid of a labeled polypeptide comprising one or more labeled aminoacids.

In some embodiments, a luminescent label may comprise a first and secondchromophore. In some embodiments, an excited state of the firstchromophore is capable of relaxation via an energy transfer to thesecond chromophore. In some embodiments, the energy transfer is aFørster resonance energy transfer (FRET). Such a FRET pair may be usefulfor providing a luminescent label with properties that make the labeleasier to differentiate from amongst a plurality of luminescent labelsin a mixture—e.g., as illustrated and described herein for labeledaptamer 106 of FIG. 1C. In yet other embodiments, a FRET pair comprisesa first chromophore of a first luminescent label and a secondchromophore of a second luminescent label—e.g., as illustrated anddescribed herein for sequencing of labeled peptides using a labeledcofactor (see, e.g., FIG. 9). In certain embodiments, the FRET pair mayabsorb excitation energy in a first spectral range and emit luminescencein a second spectral range.

In some embodiments, a luminescent label refers to a fluorophore or adye. Typically, a luminescent label comprises an aromatic orheteroaromatic compound and can be a pyrene, anthracene, naphthalene,naphthylamine, acridine, stilbene, indole, benzindole, oxazole,carbazole, thiazole, benzothiazole, benzoxazole, phenanthridine,phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine,carbocyanine, salicylate, anthranilate, coumarin, fluoroscein,rhodamine, xanthene, or other like compound.

In some embodiments, a luminescent label comprises a dye selected fromone or more of the following: 5/6-Carboxyrhodamine 6G,5-Carboxyrhodamine 6G, 6-Carboxyrhodamine 6G, 6-TAMRA, Abberior® STAR440SXP, Abberior® STAR 470SXP, Abberior® STAR 488, Abberior® STAR 512,Abberior® STAR 520SXP, Abberior® STAR 580, Abberior® STAR 600, Abberior®STAR 635, Abberior® STAR 635P, Abberior® STAR RED, Alexa Fluor® 350,Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 480, Alexa Fluor® 488,Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610-X, Alexa Fluor®633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor®700, Alexa Fluor® 750, Alexa Fluor® 790, AMCA, ATTO 390, ATTO 425, ATTO465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 542, ATTO550, ATTO 565, ATTO 590, ATTO 610, ATTO 620, ATTO 633, ATTO 647, ATTO647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, ATTOOxa12, ATTO Rho101, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14, ATTORho3B, ATTO Rho6G, ATTO Thio12, BD Horizon™ V450, BODIPY® 493/501,BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 576/589,BODIPY® 581/591, BODIPY® 630/650, BODIPY® 650/665, BODIPY® FL, BODIPY®FL-X, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, CAL Fluor® Gold 540, CALFluor® Green 510, CAL Fluor® Orange 560, CAL Fluor® Red 590, CAL Fluor®Red 610, CAL Fluor® Red 615, CAL Fluor® Red 635, Cascade® Blue, CF™350,CF™405M, CF™405S, CF™488A, CF™514, CF™532, CF™543, CF™546, CF™555,CF™568, CF™594, CF™620R, CF™633, CF™633-V1, CF™640R, CF™640R-V1,CF™640R-V2, CF™660C, CF™660R, CF™680, CF™680R, CF™680R-V1, CF™750,CF™770, CF™790, Chromeo™ 642, Chromis 425N, Chromis 500N, Chromis 515N,Chromis 530N, Chromis 550A, Chromis 550C, Chromis 550Z, Chromis 560N,Chromis 570N, Chromis 577N, Chromis 600N, Chromis 630N, Chromis 645A,Chromis 645C, Chromis 645Z, Chromis 678A, Chromis 678C, Chromis 678Z,Chromis 770A, Chromis 770C, Chromis 800A, Chromis 800C, Chromis 830A,Chromis 830C, Cy®3, Cy®3.5, Cy®3B, Cy®5, Cy®5.5, Cy®7, DyLight® 350,DyLight® 405, DyLight® 415-Col, DyLight® 425Q, DyLight® 485-LS, DyLight®488, DyLight® 504Q, DyLight® 510-LS, DyLight® 515-LS, DyLight® 521-LS,DyLight® 530-R2, DyLight® 543Q, DyLight® 550, DyLight® 554-R0, DyLight®554-R1, DyLight® 590-R2, DyLight® 594, DyLight® 610-B1, DyLight® 615-B2,DyLight® 633, DyLight® 633-B1, DyLight® 633-B2, DyLight® 650, DyLight®655-B1, DyLight® 655-B2, DyLight® 655-B3, DyLight® 655-B4, DyLight®662Q, DyLight® 675-B1, DyLight® 675-B2, DyLight® 675-B3, DyLight®675-B4, DyLight® 679-05, DyLight® 680, DyLight® 683Q, DyLight® 690-B1,DyLight® 690-B2, DyLight® 696Q, DyLight® 700-B1, DyLight® 700-B1,DyLight® 730-B1, DyLight® 730-B2, DyLight® 730-B3, DyLight® 730-B4,DyLight® 747, DyLight® 747-B1, DyLight® 747-B2, DyLight® 747-B3,DyLight® 747-B4, DyLight® 755, DyLight® 766Q, DyLight® 775-B2, DyLight®775-B3, DyLight® 775-B4, DyLight® 780-B1, DyLight® 780-B2, DyLight®780-B3, DyLight® 800, DyLight® 830-B2, Dyomics-350, Dyomics-350XL,Dyomics-360XL, Dyomics-370XL, Dyomics-375XL, Dyomics-380XL,Dyomics-390XL, Dyomics-405, Dyomics-415, Dyomics-430, Dyomics-431,Dyomics-478, Dyomics-480XL, Dyomics-481XL, Dyomics-485XL, Dyomics-490,Dyomics-495, Dyomics-505, Dyomics-510XL, Dyomics-511XL, Dyomics-520XL,Dyomics-521XL, Dyomics-530, Dyomics-547, Dyomics-547P1, Dyomics-548,Dyomics-549, Dyomics-549P1, Dyomics-550, Dyomics-554, Dyomics-555,Dyomics-556, Dyomics-560, Dyomics-590, Dyomics-591, Dyomics-594,Dyomics-601XL, Dyomics-605, Dyomics-610, Dyomics-615, Dyomics-630,Dyomics-631, Dyomics-632, Dyomics-633, Dyomics-634, Dyomics-635,Dyomics-636, Dyomics-647, Dyomics-647P1, Dyomics-648, Dyomics-648P1,Dyomics-649, Dyomics-649P1, Dyomics-650, Dyomics-651, Dyomics-652,Dyomics-654, Dyomics-675, Dyomics-676, Dyomics-677, Dyomics-678,Dyomics-679P1, Dyomics-680, Dyomics-681, Dyomics-682, Dyomics-700,Dyomics-701, Dyomics-703, Dyomics-704, Dyomics-730, Dyomics-731,Dyomics-732, Dyomics-734, Dyomics-749, Dyomics-749P1, Dyomics-750,Dyomics-751, Dyomics-752, Dyomics-754, Dyomics-776, Dyomics-777,Dyomics-778, Dyomics-780, Dyomics-781, Dyomics-782, Dyomics-800,Dyomics-831, eFluor® 450, Eosin, FITC, Fluorescein, HiLyte™ Fluor 405,HiLyte™ Fluor 488, HiLyte™ Fluor 532, HiLyte™ Fluor 555, HiLyte™ Fluor594, HiLyte™ Fluor 647, HiLyte™ Fluor 680, HiLyte™ Fluor 750, IRDye®680LT, IRDye® 750, IRDye® 800CW, JOE, LightCycler® 640R, LightCycler®Red 610, LightCycler® Red 640, LightCycler® Red 670, LightCycler® Red705, Lissamine Rhodamine B, Napthofluorescein, Oregon Green® 488, OregonGreen® 514, Pacific Blue™ Pacific Green™, Pacific Orange™, PET, PF350,PF405, PF415, PF488, PF505, PF532, PF546, PF555P, PF568, PF594, PF610,PF633P, PF647P, Quasar® 570, Quasar® 670, Quasar® 705, Rhodamine 123,Rhodamine 6G, Rhodamine B, Rhodamine Green, Rhodamine Green-X, RhodamineRed, ROX, Seta™ 375, Seta™ 470, Seta™ 555, Seta™ 632, Seta™ 633, Seta™650, Seta™ 660, Seta™ 670, Seta™ 680, Seta™ 700, Seta™ 750, Seta™ 780,Seta™ APC-780, Seta™ PerCP-680, Seta™ R-PE-670, Seta™ 646, SeTau 380,SeTau 425, SeTau 647, SeTau 405, Square 635, Square 650, Square 660,Square 672, Square 680, Sulforhodamine 101, TAMRA, TET, Texas Red®, TMR,TRITC, Yakima Yellow™ Zenon®, Zy3, Zy5, Zy5.5, and Zy7.

Luminescence

In some aspects, the application relates to polypeptide sequencingand/or identification based on one or more luminescence properties of aluminescent label. In some embodiments, a luminescent label isidentified based on luminescence lifetime, luminescence intensity,brightness, absorption spectra, emission spectra, luminescence quantumyield, or a combination of two or more thereof. In some embodiments, aplurality of types of luminescent labels can be distinguished from eachother based on different luminescence lifetimes, luminescenceintensities, brightnesses, absorption spectra, emission spectra,luminescence quantum yields, or combinations of two or more thereof.Identifying may mean assigning the exact identity and/or quantity of onetype of amino acid (e.g., a single type or a subset of types) associatedwith a luminescent label, and may also mean assigning an amino acidlocation in a polypeptide relative to other types of amino acids.

In some embodiments, luminescence is detected by exposing a luminescentlabel to a series of separate light pulses and evaluating the timing orother properties of each photon that is emitted from the label. In someembodiments, information for a plurality of photons emitted sequentiallyfrom a label is aggregated and evaluated to identify the label andthereby identify an associated type of amino acid. In some embodiments,a luminescence lifetime of a label is determined from a plurality ofphotons that are emitted sequentially from the label, and theluminescence lifetime can be used to identify the label. In someembodiments, a luminescence intensity of a label is determined from aplurality of photons that are emitted sequentially from the label, andthe luminescence intensity can be used to identify the label. In someembodiments, a luminescence lifetime and luminescence intensity of alabel is determined from a plurality of photons that are emittedsequentially from the label, and the luminescence lifetime andluminescence intensity can be used to identify the label.

In some aspects of the application, a single polypeptide molecule isexposed to a plurality of separate light pulses and a series of emittedphotons are detected and analyzed. In some embodiments, the series ofemitted photons provides information about the single polypeptidemolecule that is present and that does not change in the reaction sampleover the time of the experiment. However, in some embodiments, theseries of emitted photons provides information about a series ofdifferent molecules that are present at different times in the reactionsample (e.g., as a reaction or process progresses). By way of exampleand not limitation, such information may be used to sequence and/oridentify a polypeptide subjected to chemical or enzymatic degradation inaccordance with the application.

In certain embodiments, a luminescent label absorbs one photon and emitsone photon after a time duration. In some embodiments, the luminescencelifetime of a label can be determined or estimated by measuring the timeduration. In some embodiments, the luminescence lifetime of a label canbe determined or estimated by measuring a plurality of time durationsfor multiple pulse events and emission events. In some embodiments, theluminescence lifetime of a label can be differentiated amongst theluminescence lifetimes of a plurality of types of labels by measuringthe time duration. In some embodiments, the luminescence lifetime of alabel can be differentiated amongst the luminescence lifetimes of aplurality of types of labels by measuring a plurality of time durationsfor multiple pulse events and emission events. In certain embodiments, alabel is identified or differentiated amongst a plurality of types oflabels by determining or estimating the luminescence lifetime of thelabel. In certain embodiments, a label is identified or differentiatedamongst a plurality of types of labels by differentiating theluminescence lifetime of the label amongst a plurality of theluminescence lifetimes of a plurality of types of labels.

Determination of a luminescence lifetime of a luminescent label can beperformed using any suitable method (e.g., by measuring the lifetimeusing a suitable technique or by determining time-dependentcharacteristics of emission). In some embodiments, determining theluminescence lifetime of one label comprises determining the lifetimerelative to another label. In some embodiments, determining theluminescence lifetime of a label comprises determining the lifetimerelative to a reference. In some embodiments, determining theluminescence lifetime of a label comprises measuring the lifetime (e.g.,fluorescence lifetime). In some embodiments, determining theluminescence lifetime of a label comprises determining one or moretemporal characteristics that are indicative of lifetime. In someembodiments, the luminescence lifetime of a label can be determinedbased on a distribution of a plurality of emission events (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 60, 70, 80, 90, 100, or more emission events) occurring across oneor more time-gated windows relative to an excitation pulse. For example,a luminescence lifetime of a label can be distinguished from a pluralityof labels having different luminescence lifetimes based on thedistribution of photon arrival times measured with respect to anexcitation pulse.

It should be appreciated that a luminescence lifetime of a luminescentlabel is indicative of the timing of photons emitted after the labelreaches an excited state and the label can be distinguished byinformation indicative of the timing of the photons. Some embodimentsmay include distinguishing a label from a plurality of labels based onthe luminescence lifetime of the label by measuring times associatedwith photons emitted by the label. The distribution of times may providean indication of the luminescence lifetime which may be determined fromthe distribution. In some embodiments, the label is distinguishable fromthe plurality of labels based on the distribution of times, such as bycomparing the distribution of times to a reference distributioncorresponding to a known label. In some embodiments, a value for theluminescence lifetime is determined from the distribution of times.

As used herein, in some embodiments, luminescence intensity refers tothe number of emitted photons per unit time that are emitted by aluminescent label which is being excited by delivery of a pulsedexcitation energy. In some embodiments, the luminescence intensityrefers to the detected number of emitted photons per unit time that areemitted by a label which is being excited by delivery of a pulsedexcitation energy, and are detected by a particular sensor or set ofsensors.

As used herein, in some embodiments, brightness refers to a parameterthat reports on the average emission intensity per luminescent label.Thus, in some embodiments, “emission intensity” may be used to generallyrefer to brightness of a composition comprising one or more labels. Insome embodiments, brightness of a label is equal to the product of itsquantum yield and extinction coefficient.

As used herein, in some embodiments, luminescence quantum yield refersto the fraction of excitation events at a given wavelength or within agiven spectral range that lead to an emission event, and is typicallyless than 1. In some embodiments, the luminescence quantum yield of aluminescent label described herein is between 0 and about 0.001, betweenabout 0.001 and about 0.01, between about 0.01 and about 0.1, betweenabout 0.1 and about 0.5, between about 0.5 and 0.9, or between about 0.9and 1. In some embodiments, a label is identified by determining orestimating the luminescence quantum yield.

As used herein, in some embodiments, an excitation energy is a pulse oflight from a light source. In some embodiments, an excitation energy isin the visible spectrum. In some embodiments, an excitation energy is inthe ultraviolet spectrum. In some embodiments, an excitation energy isin the infrared spectrum. In some embodiments, an excitation energy isat or near the absorption maximum of a luminescent label from which aplurality of emitted photons are to be detected. In certain embodiments,the excitation energy is between about 500 nm and about 700 nm (e.g.,between about 500 nm and about 600 nm, between about 600 nm and about700 nm, between about 500 nm and about 550 nm, between about 550 nm andabout 600 nm, between about 600 nm and about 650 nm, or between about650 nm and about 700 nm). In certain embodiments, an excitation energymay be monochromatic or confined to a spectral range. In someembodiments, a spectral range has a range of between about 0.1 nm andabout 1 nm, between about 1 nm and about 2 nm, or between about 2 nm andabout 5 nm. In some embodiments, a spectral range has a range of betweenabout 5 nm and about 10 nm, between about 10 nm and about 50 nm, orbetween about 50 nm and about 100 nm.

Sequencing

Aspects of the application relate to sequencing biological polymers,such as polypeptides and proteins. As used herein, “sequencing,”“sequence determination,” “determining a sequence,” and like terms, inreference to a polypeptide or protein includes determination of partialsequence information as well as full sequence information of thepolypeptide or protein. That is, the terminology includes sequencecomparisons, fingerprinting, probabalistic fingerprinting, and likelevels of information about a target molecule, as well as the expressidentification and ordering of each amino acid of the target moleculewithin a region of interest. In some embodiments, the terminologyincludes identifying a single amino acid of a polypeptide. In yet otherembodiments, more than one amino acid of a polypeptide is identified. Asused herein, in some embodiments, “identifying,” “determining theidentity,” and like terms, in reference to an amino acid includesdetermination of an express identity of an amino acid as well asdetermination of a probability of an express identity of an amino acid.For example, in some embodiments, an amino acid is identified bydetermining a probability (e.g., from 0% to 100%) that the amino acid isof a specific type, or by determining a probability for each of aplurality of specific types. Accordingly, in some embodiments, the terms“amino acid sequence,” “polypeptide sequence,” and “protein sequence” asused herein may refer to the polypeptide or protein material itself andis not restricted to the specific sequence information (e.g., thesuccession of letters representing the order of amino acids from oneterminus to another terminus) that biochemically characterizes aspecific polypeptide or protein.

In some embodiments, sequencing of a polypeptide molecule comprisesidentifying at least two (e.g., at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, or more) amino acidsin the polypeptide molecule. In some embodiments, the at least two aminoacids are contiguous amino acids. In some embodiments, the at least twoamino acids are non-contiguous amino acids.

In some embodiments, sequencing of a polypeptide molecule comprisesidentification of less than 100% (e.g., less than 99%, less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, less than 50%, lessthan 45%, less than 40%, less than 35%, less than 30%, less than 25%,less than 20%, less than 15%, less than 10%, less than 5%, less than 1%or less) of all amino acids in the polypeptide molecule. For example, insome embodiments, sequencing of a polypeptide molecule comprisesidentification of less than 100% of one type of amino acid in thepolypeptide molecule (e.g., identification of a portion of all aminoacids of one type in the polypeptide molecule). In some embodiments,sequencing of a polypeptide molecule comprises identification of lessthan 100% of each type of amino acid in the polypeptide molecule.

In some embodiments, sequencing of a polypeptide molecule comprisesidentification of at least 1, at least 5, at least 10, at least 15, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 55, at least 60, at least 65, at least 70, atleast 75, at least 80, at least 85, at least 90, at least 95, at least100 or more types of amino acids in the polypeptide.

In some embodiments, the application provides compositions and methodsfor sequencing a polypeptide by identifying a series of amino acids thatare present at a terminus of a polypeptide over time (e.g., by iterativedetection and cleavage of amino acids at the terminus). In yet otherembodiments, the application provides compositions and methods forsequencing a polypeptide by identifying labeled amino content of thepolypeptide and comparing to a reference sequence database.

In some embodiments, the application provides compositions and methodsfor sequencing a polypeptide by sequencing a plurality of fragments ofthe polypeptide. In some embodiments, sequencing a polypeptide comprisescombining sequence information for a plurality of polypeptide fragmentsto identify and/or determine a sequence for the polypeptide. In someembodiments, combining sequence information may be performed by computerhardware and software. The methods described herein may allow for a setof related polypeptides, such as an entire proteome of an organism, tobe sequenced. In some embodiments, a plurality of single moleculesequencing reactions are performed in parallel (e.g., on a single chip)according to aspects of the present application. For example, in someembodiments, a plurality of single molecule sequencing reactions areeach performed in separate sample wells on a single chip.

In some embodiments, methods provided herein may be used for thesequencing and identification of an individual protein in a samplecomprising a complex mixture of proteins. In some embodiments, theapplication provides methods of uniquely identifying an individualprotein in a complex mixture of proteins. In some embodiments, anindividual protein is detected in a mixed sample by determining apartial amino acid sequence of the protein. In some embodiments, thepartial amino acid sequence of the protein is within a contiguousstretch of approximately 5 to 50 amino acids.

Without wishing to be bound by any particular theory, it is believedthat most human proteins can be identified using incomplete sequenceinformation with reference to proteomic databases. For example, simplemodeling of the human proteome has shown that approximately 98% ofproteins can be uniquely identified by detecting just four types ofamino acids within a stretch of 6 to 40 amino acids (see, e.g.,Swaminathan, et al. PLoS Comput Biol. 2015, 11(2):e1004080; and Yao, etal. Phys. Biol. 2015, 12(5):055003). Therefore, a complex mixture ofproteins can be degraded (e.g., chemically degraded, enzymaticallydegraded) into short polypeptide fragments of approximately 6 to 40amino acids, and sequencing of this polypeptide library would reveal theidentity and abundance of each of the proteins present in the originalcomplex mixture. Compositions and methods for selective amino acidlabeling and identifying polypeptides by determining partial sequenceinformation are described in detail in U.S. patent application Ser. No.15/510,962, filed Sep. 15, 2015, titled “SINGLE MOLECULE PEPTIDESEQUENCING,” which is incorporated by reference in its entirety.

Embodiments are capable of sequencing single polypeptide molecules withhigh accuracy, such as an accuracy of at least about 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or99.9999%. In some embodiments, the target molecule used in singlemolecule sequencing is a polypeptide that is immobilized to a surface ofa solid support such as a bottom surface or a sidewall surface of asample well. The sample well also can contain any other reagents neededfor a sequencing reaction in accordance with the application, such asone or more suitable buffers, co-factors, labeled affinity reagents, andenzymes (e.g., catalytically active or inactive exopeptidase enzymes,which may be luminescently labeled or unlabeled).

As described above, in some embodiments, sequencing in accordance withthe application comprises identifying an amino acid by determining aprobability that the amino acid is of a specific type. Conventionalprotein identification systems require identification of each amino acidin a polypeptide to identify the polypeptide. However, it is difficultto accurately identify each amino acid in a polypeptide. For example,data collected from an interaction in which a first recognition moleculeassociates with a first amino acid may not be sufficiently differentfrom data collected from an interaction in which a second recognitionmolecule associates with a second amino acid to differentiate betweenthe two amino acids. In some embodiments, sequencing in accordance withthe application avoids this problem by using a protein identificationsystem that, unlike conventional protein identification systems, doesnot require (but does not preclude) identification of each amino acid inthe protein.

Accordingly, in some embodiments, sequencing in accordance with theapplication may be carried out using a protein identification systemthat uses machine learning techniques to identify proteins. In someembodiments, the system operates by: (1) collecting data about apolypeptide of a protein using a real-time protein sequencing device;(2) using a machine learning model and the collected data to identifyprobabilities that certain amino acids are part of the polypeptide atrespective locations; and (3) using the identified probabilities, as a“probabilistic fingerprint” to identify the protein. In someembodiments, data about the polypeptide of the protein may be obtainedusing reagents that selectively bind amino acids. As an example, thereagents and/or amino acids may be labeled with luminescent labels thatemit light in response to application of excitation energy. In thisexample, a protein sequencing device may apply excitation energy to asample of a protein (e.g., a polypeptide) during binding interactions ofreagents with amino acids in the sample. In some embodiments, one ormore sensors in the sequencing device (e.g., a photodetector, anelectrical sensor, and/or any other suitable type of sensor) may detectbinding interactions. In turn, the data collected and/or derived fromthe detected light emissions may be provided to the machine learningmodel. Machine learning models and associated systems and methods aredescribed in detail in U.S. Provisional Patent Appl. No. 62/860,750,filed Jun. 12, 2019, titled “MACHINE LEARNING ENABLED PROTEINIDENTIFICATION,” which is incorporated by reference in its entirety.

Sequencing in accordance with the application, in some aspects, mayinvolve immobilizing a polypeptide on a surface of a substrate (e.g., ofa solid support, for example a chip, for example an integrated device asdescribed herein). In some embodiments, a polypeptide may be immobilizedon a surface of a sample well (e.g., on a bottom surface of a samplewell) on a substrate. In some embodiments, the N-terminal amino acid ofthe polypeptide is immobilized (e.g., attached to the surface). In someembodiments, the C-terminal amino acid of the polypeptide is immobilized(e.g., attached to the surface). In some embodiments, one or morenon-terminal amino acids are immobilized (e.g., attached to thesurface). The immobilized amino acid(s) can be attached using anysuitable covalent or non-covalent linkage, for example as described inthis application. In some embodiments, a plurality of polypeptides areattached to a plurality of sample wells (e.g., with one polypeptideattached to a surface, for example a bottom surface, of each samplewell), for example in an array of sample wells on a substrate.

Sequencing in accordance with the application, in some aspects, may beperformed using a system that permits single molecule analysis. Thesystem may include an integrated device and an instrument configured tointerface with the integrated device. The integrated device may includean array of pixels, where individual pixels include a sample well and atleast one photodetector. The sample wells of the integrated device maybe formed on or through a surface of the integrated device and beconfigured to receive a sample placed on the surface of the integrateddevice. Collectively, the sample wells may be considered as an array ofsample wells. The plurality of sample wells may have a suitable size andshape such that at least a portion of the sample wells receive a singlesample (e.g., a single molecule, such as a polypeptide). In someembodiments, the number of samples within a sample well may bedistributed among the sample wells of the integrated device such thatsome sample wells contain one sample while others contain zero, two ormore samples.

Excitation light is provided to the integrated device from one or morelight source external to the integrated device. Optical components ofthe integrated device may receive the excitation light from the lightsource and direct the light towards the array of sample wells of theintegrated device and illuminate an illumination region within thesample well. In some embodiments, a sample well may have a configurationthat allows for the sample to be retained in proximity to a surface ofthe sample well, which may ease delivery of excitation light to thesample and detection of emission light from the sample. A samplepositioned within the illumination region may emit emission light inresponse to being illuminated by the excitation light. For example, thesample may be labeled with a fluorescent marker, which emits light inresponse to achieving an excited state through the illumination ofexcitation light. Emission light emitted by a sample may then bedetected by one or more photodetectors within a pixel corresponding tothe sample well with the sample being analyzed. When performed acrossthe array of sample wells, which may range in number betweenapproximately 10,000 pixels to 1,000,000 pixels according to someembodiments, multiple samples can be analyzed in parallel.

The integrated device may include an optical system for receivingexcitation light and directing the excitation light among the samplewell array. The optical system may include one or more grating couplersconfigured to couple excitation light to the integrated device anddirect the excitation light to other optical components. The opticalsystem may include optical components that direct the excitation lightfrom a grating coupler towards the sample well array. Such opticalcomponents may include optical splitters, optical combiners, andwaveguides. In some embodiments, one or more optical splitters maycouple excitation light from a grating coupler and deliver excitationlight to at least one of the waveguides. According to some embodiments,the optical splitter may have a configuration that allows for deliveryof excitation light to be substantially uniform across all thewaveguides such that each of the waveguides receives a substantiallysimilar amount of excitation light. Such embodiments may improveperformance of the integrated device by improving the uniformity ofexcitation light received by sample wells of the integrated device.Examples of suitable components, e.g., for coupling excitation light toa sample well and/or directing emission light to a photodetector, toinclude in an integrated device are described in U.S. patent applicationSer. No. 14/821,688, filed Aug. 7, 2015, titled “INTEGRATED DEVICE FORPROBING, DETECTING AND ANALYZING MOLECULES,” and U.S. patent applicationSer. No. 14/543,865, filed Nov. 17, 2014, titled “INTEGRATED DEVICE WITHEXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZING MOLECULES,”both of which are incorporated by reference in their entirety. Examplesof suitable grating couplers and waveguides that may be implemented inthe integrated device are described in U.S. patent application Ser. No.15/844,403, filed Dec. 15, 2017, titled “OPTICAL COUPLER AND WAVEGUIDESYSTEM,” which is incorporated by reference in its entirety.

Additional photonic structures may be positioned between the samplewells and the photodetectors and configured to reduce or preventexcitation light from reaching the photodetectors, which may otherwisecontribute to signal noise in detecting emission light. In someembodiments, metal layers which may act as a circuitry for theintegrated device, may also act as a spatial filter. Examples ofsuitable photonic structures may include spectral filters, apolarization filters, and spatial filters and are described in U.S.patent application Ser. No. 16/042,968, filed Jul. 23, 2018, titled“OPTICAL REJECTION PHOTONIC STRUCTURES,” which is incorporated byreference in its entirety.

Components located off of the integrated device may be used to positionand align an excitation source to the integrated device. Such componentsmay include optical components including lenses, mirrors, prisms,windows, apertures, attenuators, and/or optical fibers. Additionalmechanical components may be included in the instrument to allow forcontrol of one or more alignment components. Such mechanical componentsmay include actuators, stepper motors, and/or knobs. Examples ofsuitable excitation sources and alignment mechanisms are described inU.S. patent application Ser. No. 15/161,088, filed May 20, 2016, titled“PULSED LASER AND SYSTEM,” which is incorporated by reference in itsentirety. Another example of a beam-steering module is described in U.S.patent application Ser. No. 15/842,720, filed Dec. 14, 2017, titled“COMPACT BEAM SHAPING AND STEERING ASSEMBLY,” which is incorporatedherein by reference. Additional examples of suitable excitation sourcesare described in U.S. patent application Ser. No. 14/821,688, filed Aug.7, 2015, titled “INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZINGMOLECULES,” which is incorporated by reference in its entirety.

The photodetector(s) positioned with individual pixels of the integrateddevice may be configured and positioned to detect emission light fromthe pixel's corresponding sample well. Examples of suitablephotodetectors are described in U.S. patent application Ser. No.14/821,656, filed Aug. 7, 2015, titled “INTEGRATED DEVICE FOR TEMPORALBINNING OF RECEIVED PHOTONS,” which is incorporated by reference in itsentirety. In some embodiments, a sample well and its respectivephotodetector(s) may be aligned along a common axis. In this manner, thephotodetector(s) may overlap with the sample well within the pixel.

Characteristics of the detected emission light may provide an indicationfor identifying the marker associated with the emission light. Suchcharacteristics may include any suitable type of characteristic,including an arrival time of photons detected by a photodetector, anamount of photons accumulated over time by a photodetector, and/or adistribution of photons across two or more photodetectors. In someembodiments, a photodetector may have a configuration that allows forthe detection of one or more timing characteristics associated with asample's emission light (e.g., luminescence lifetime). The photodetectormay detect a distribution of photon arrival times after a pulse ofexcitation light propagates through the integrated device, and thedistribution of arrival times may provide an indication of a timingcharacteristic of the sample's emission light (e.g., a proxy forluminescence lifetime). In some embodiments, the one or morephotodetectors provide an indication of the probability of emissionlight emitted by the marker (e.g., luminescence intensity). In someembodiments, a plurality of photodetectors may be sized and arranged tocapture a spatial distribution of the emission light. Output signalsfrom the one or more photodetectors may then be used to distinguish amarker from among a plurality of markers, where the plurality of markersmay be used to identify a sample within the sample. In some embodiments,a sample may be excited by multiple excitation energies, and emissionlight and/or timing characteristics of the emission light emitted by thesample in response to the multiple excitation energies may distinguish amarker from a plurality of markers.

In operation, parallel analyses of samples within the sample wells arecarried out by exciting some or all of the samples within the wellsusing excitation light and detecting signals from sample emission withthe photodetectors. Emission light from a sample may be detected by acorresponding photodetector and converted to at least one electricalsignal. The electrical signals may be transmitted along conducting linesin the circuitry of the integrated device, which may be connected to aninstrument interfaced with the integrated device. The electrical signalsmay be subsequently processed and/or analyzed. Processing or analyzingof electrical signals may occur on a suitable computing device eitherlocated on or off the instrument.

The instrument may include a user interface for controlling operation ofthe instrument and/or the integrated device. The user interface may beconfigured to allow a user to input information into the instrument,such as commands and/or settings used to control the functioning of theinstrument. In some embodiments, the user interface may include buttons,switches, dials, and a microphone for voice commands. The user interfacemay allow a user to receive feedback on the performance of theinstrument and/or integrated device, such as proper alignment and/orinformation obtained by readout signals from the photodetectors on theintegrated device. In some embodiments, the user interface may providefeedback using a speaker to provide audible feedback. In someembodiments, the user interface may include indicator lights and/or adisplay screen for providing visual feedback to a user.

In some embodiments, the instrument may include a computer interfaceconfigured to connect with a computing device. The computer interfacemay be a USB interface, a FireWire interface, or any other suitablecomputer interface. A computing device may be any general purposecomputer, such as a laptop or desktop computer. In some embodiments, acomputing device may be a server (e.g., cloud-based server) accessibleover a wireless network via a suitable computer interface. The computerinterface may facilitate communication of information between theinstrument and the computing device. Input information for controllingand/or configuring the instrument may be provided to the computingdevice and transmitted to the instrument via the computer interface.Output information generated by the instrument may be received by thecomputing device via the computer interface. Output information mayinclude feedback about performance of the instrument, performance of theintegrated device, and/or data generated from the readout signals of thephotodetector.

In some embodiments, the instrument may include a processing deviceconfigured to analyze data received from one or more photodetectors ofthe integrated device and/or transmit control signals to the excitationsource(s). In some embodiments, the processing device may comprise ageneral purpose processor, a specially-adapted processor (e.g., acentral processing unit (CPU) such as one or more microprocessor ormicrocontroller cores, a field-programmable gate array (FPGA), anapplication-specific integrated circuit (ASIC), a custom integratedcircuit, a digital signal processor (DSP), or a combination thereof). Insome embodiments, the processing of data from one or more photodetectorsmay be performed by both a processing device of the instrument and anexternal computing device. In other embodiments, an external computingdevice may be omitted and processing of data from one or morephotodetectors may be performed solely by a processing device of theintegrated device.

According to some embodiments, the instrument that is configured toanalyze samples based on luminescence emission characteristics maydetect differences in luminescence lifetimes and/or intensities betweendifferent luminescent molecules, and/or differences between lifetimesand/or intensities of the same luminescent molecules in differentenvironments. The inventors have recognized and appreciated thatdifferences in luminescence emission lifetimes can be used to discernbetween the presence or absence of different luminescent moleculesand/or to discern between different environments or conditions to whicha luminescent molecule is subjected. In some cases, discerningluminescent molecules based on lifetime (rather than emissionwavelength, for example) can simplify aspects of the system. As anexample, wavelength-discriminating optics (such as wavelength filters,dedicated detectors for each wavelength, dedicated pulsed opticalsources at different wavelengths, and/or diffractive optics) may bereduced in number or eliminated when discerning luminescent moleculesbased on lifetime. In some cases, a single pulsed optical sourceoperating at a single characteristic wavelength may be used to excitedifferent luminescent molecules that emit within a same wavelengthregion of the optical spectrum but have measurably different lifetimes.An analytic system that uses a single pulsed optical source, rather thanmultiple sources operating at different wavelengths, to excite anddiscern different luminescent molecules emitting in a same wavelengthregion can be less complex to operate and maintain, more compact, andmay be manufactured at lower cost.

Although analytic systems based on luminescence lifetime analysis mayhave certain benefits, the amount of information obtained by an analyticsystem and/or detection accuracy may be increased by allowing foradditional detection techniques. For example, some embodiments of thesystems may additionally be configured to discern one or more propertiesof a sample based on luminescence wavelength and/or luminescenceintensity. In some implementations, luminescence intensity may be usedadditionally or alternatively to distinguish between differentluminescent labels. For example, some luminescent labels may emit atsignificantly different intensities or have a significant difference intheir probabilities of excitation (e.g., at least a difference of about35%) even though their decay rates may be similar. By referencing binnedsignals to measured excitation light, it may be possible to distinguishdifferent luminescent labels based on intensity levels.

According to some embodiments, different luminescence lifetimes may bedistinguished with a photodetector that is configured to time-binluminescence emission events following excitation of a luminescentlabel. The time binning may occur during a single charge-accumulationcycle for the photodetector. A charge-accumulation cycle is an intervalbetween read-out events during which photo-generated carriers areaccumulated in bins of the time-binning photodetector. Examples of atime-binning photodetector are described in U.S. patent application Ser.No. 14/821,656, filed Aug. 7, 2015, titled “INTEGRATED DEVICE FORTEMPORAL BINNING OF RECEIVED PHOTONS,” which is incorporated herein byreference. In some embodiments, a time-binning photodetector maygenerate charge carriers in a photon absorption/carrier generationregion and directly transfer charge carriers to a charge carrier storagebin in a charge carrier storage region. In such embodiments, thetime-binning photodetector may not include a carrier travel/captureregion. Such a time-binning photodetector may be referred to as a“direct binning pixel.” Examples of time-binning photodetectors,including direct binning pixels, are described in U.S. patentapplication Ser. No. 15/852,571, filed Dec. 22, 2017, titled “INTEGRATEDPHOTODETECTOR WITH DIRECT BINNING PIXEL,” which is incorporated hereinby reference.

In some embodiments, different numbers of fluorophores of the same typemay be linked to different reagents in a sample, so that each reagentmay be identified based on luminescence intensity. For example, twofluorophores may be linked to a first labeled affinity reagent and fouror more fluorophores may be linked to a second labeled affinity reagent.Because of the different numbers of fluorophores, there may be differentexcitation and fluorophore emission probabilities associated with thedifferent affinity reagents. For example, there may be more emissionevents for the second labeled affinity reagent during a signalaccumulation interval, so that the apparent intensity of the bins issignificantly higher than for the first labeled affinity reagent.

The inventors have recognized and appreciated that distinguishingnucleotides or any other biological or chemical samples based onfluorophore decay rates and/or fluorophore intensities may enable asimplification of the optical excitation and detection systems. Forexample, optical excitation may be performed with a single-wavelengthsource (e.g., a source producing one characteristic wavelength ratherthan multiple sources or a source operating at multiple differentcharacteristic wavelengths). Additionally, wavelength discriminatingoptics and filters may not be needed in the detection system. Also, asingle photodetector may be used for each sample well to detect emissionfrom different fluorophores. The phrase “characteristic wavelength” or“wavelength” is used to refer to a central or predominant wavelengthwithin a limited bandwidth of radiation (e.g., a central or peakwavelength within a 20 nm bandwidth output by a pulsed optical source).In some cases, “characteristic wavelength” or “wavelength” may be usedto refer to a peak wavelength within a total bandwidth of radiationoutput by a source.

Computational Techniques

Aspects of the present application relate to computational techniquesfor analyzing the data generated by the polypeptide sequencingtechniques described herein. As discussed above, for example inconnection with FIGS. 1A and 1B, the data generated by using thesesequencing techniques may include a series of signal pulses indicativeof instances where an amino acid recognition molecule is associated withan amino acid exposed at the terminus of the polypeptide beingsequenced. The series of signal pulses may have varying one or morefeatures (e.g., pulse duration, interpulse duration, change inmagnitude), depending on the type of amino acid presently at theterminus, over time as the degradation process proceeds in removingsuccessive amino acids. The resulting signal trace may includecharacteristic patterns, which arise from the varying one or morefeatures, associated with respective amino acids. The computationaltechniques described herein may be implemented as part of analyzing suchdata obtained using these sequencing techniques to identify an aminoacid sequence.

Some embodiments may involve obtaining data during a degradation processof a polypeptide, analyzing the data to determine portions of the datacorresponding to amino acids that are sequentially exposed at a terminusof the polypeptide during the degradation process, and outputting anamino acid sequence representative of the polypeptide. FIG. 11 is adiagram of an illustrative processing pipeline 1100 for identifying anamino acid sequence by analyzing data obtained using the polypeptidesequencing techniques described herein. As shown in FIG. 11, analyzingsequencing data 1102 may involve using association event identificationtechnique 1104 and amino acid identification technique 1106 to outputamino acid sequence(s) 1108.

As discussed herein, sequencing data 1102 may be obtained during adegradation process of a polypeptide. In some embodiments, thesequencing data 1102 is indicative of amino acid identity at theterminus of the polypeptide during the degradation process. In someembodiments, the sequencing data 1102 is indicative of a signal producedby one or more amino acid recognition molecules binding to differenttypes of terminal amino acids at the terminus during the degradationprocess. Exemplary sequencing data is shown in FIGS. 1A and 1B, whichare discussed above.

Depending on how signals are generated during the degradation process,sequencing data 1102 may be indicative of one or more different types ofsignals. In some embodiments, sequencing data 1102 is indicative of aluminescent signal generated during the degradation process. Forexample, a luminescent label may be used to label an amino acidrecognition molecule, and luminescence emitted by the luminescent labelmay be detected as the amino acid recognition molecule associates with aparticular amino acid, resulting in a luminescent signal. In someembodiments, sequencing data 1102 is indicative of an electrical signalgenerated during the degradation process. For example, a polypeptidemolecule being sequenced may be immobilized to a nanopore, and anelectrical signal (e.g., changes in conductance) may be detected as anamino acid recognition molecule associates with a particular amino acid.

Some embodiments involve analyzing sequencing data 1102 to determineportions of sequencing data 1102 corresponding to amino acids that aresequentially exposed at a terminus of the polypeptide during thedegradation process. As shown in FIG. 11, association eventidentification technique 1104 may access sequencing data 1102 andanalyze sequencing data to identify portions of sequencing data 1102that correspond to association events. The association events maycorrespond to characteristic patterns, such as CP₁ and CP₂ shown in FIG.1B, in the data. In some embodiments, association event identificationtechnique 1104 may involve detecting a series of cleavage events anddetermining portions of sequencing data 1102 between successive cleavageevents. As an example, a cleavage event between CP₁ and CP₂ shown inFIG. 1B may be detected such that a first portion of the datacorresponding to CP₁ may be identified as a first association event anda second portion of the data corresponding CP₂ may be identified as asecond association event.

Some embodiments involve identifying a type of amino acid for one ormore of the determined portions of sequencing data 1102. As shown inFIG. 11, amino acid identification technique 1106 may be used todetermine a type of amino acid for one or more of the association eventsidentified by association event identification technique 1104. In someembodiments, the individual portions of data identified by associationevent identification technique 1104 may include a pulse pattern, andamino acid identification technique 1106 may determine a type of aminoacid for one or more of the portions based on its respective pulsepattern. Referring to FIG. 1B, amino acid identification technique 1106may identify a first type of amino acid for CP₁ and a second type ofamino acid for CP₂. In some embodiments, determining the type of aminoacid may include identifying an amount of time within a portion of data,such as a portion identified using association event identificationtechnique 1104, when the data is above a threshold value and comparingthe amount of time to a duration of time for the portion of data. Forexample, identifying a type of amino acid for CP₁ may includedetermining an amount of time within CP₁ where the signal is above athreshold value, such as time period, pd, where the signal is aboveM_(L), and comparing it to a total duration of time for CP₁. In someembodiments, determining the type of amino acid may involve identifyingone or more pulse durations for one or more portions of data identifiedby association event identification technique 1102. For example,identifying a type of amino acid for CP₁ may include determining a pulseduration for CP₁, such as time period, pd. In some embodiments,determining the type of amino acid may involve identifying one or moreinterpulse durations for one or more portions of the data identifiedusing association event identification technique 1104. For example,identifying a type of amino acid for CP₁ may include identifying aninterpulse duration, such as ipd.

By identifying a type of amino acid for successive portions ofsequencing data 1102, amino acid identification technique 1106 mayoutput amino acid sequence(s) 1108 representative of the polypeptide. Insome embodiments, the amino acid sequence includes a series of aminoacids corresponding to the portions of data identified using associationevent identification technique 1104.

FIG. 12 is a flow chart of an illustrative process 1200 for determiningan amino acid sequence of a polypeptide molecule, in accordance withsome embodiments of the technology described herein. Process 1200 may beperformed on any suitable computing device(s) (e.g., a single computingdevice, multiple computing devices co-located in a single physicallocation or located in multiple physical locations remote from oneanother, one or more computing devices part of a cloud computing system,etc.), as aspects of the technology described herein are not limited inthis respect. In some embodiments, association event identificationtechnique 1104 and amino acid identification technique 1106 may performsome or all of process 1200 to determine amino acid sequence(s).

Process 1200 begins at act 1202, which involves contacting a singlepolypeptide molecule with one or more terminal amino acid recognitionmolecules. Next, process 1200 proceeds to act 1104, which involvesdetecting a series of signal pulses indicative of association of the oneor more terminal amino acid recognition molecules with successive aminoacids exposed at a terminus of the single polypeptide while the singlepolypeptide is being degraded. The series of pulses may allow forsequencing of the single polypeptide molecule, such as by usingassociation event identification technique 1104 and amino acididentification technique 1106.

In some embodiments, process 1200 may include act 1206, which involvesidentifying a first type of amino acid in the single polypeptidemolecule based on a first characteristic pattern in the series of signalpulses, such as by using amino acid identification technique 1106.

FIG. 13 is a flow chart of an illustrative process 1300 for determiningan amino acid sequence representative of a polypeptide, in accordancewith some embodiments of the technology described herein. Process 1300may be performed on any suitable computing device(s) (e.g., a singlecomputing device, multiple computing devices co-located in a singlephysical location or located in multiple physical locations remote fromone another, one or more computing devices part of a cloud computingsystem, etc.), as aspects of the technology described herein are notlimited in this respect. In some embodiments, association eventidentification technique 1104 and amino acid identification technique1106 may perform some or all of process 1300 to determine amino acidsequence(s).

Process 1300 begins at act 1302, where data during a degradation processof a polypeptide is obtained. In some embodiments, the data isindicative of amino acid identity at the terminus of the polypeptideduring the degradation process. In some embodiments, the data isindicative of a signal produced by one or more amino acid recognitionmolecules binding to different types of terminal amino acids at theterminus during the degradation process. In some embodiments, the datais indicative of a luminescent signal generated during the degradationprocess. In some embodiments, the data is indicative of an electricalsignal generated during the degradation process.

Next, process 1300 proceeds to act 1304, where the data is analyzed todetermine portions of the data corresponding to amino acids that aresequentially exposed at a terminus of the polypeptide during thedegradation process, such as by using association event identificationtechnique 1104 and amino acid identification technique 1106. In someembodiments, analyzing the data further comprises detecting a series ofcleavage events and determining the portions of the data betweensuccessive cleavage events, such as by using association eventidentification technique 1104.

In some embodiments, analyzing the data further comprises determining atype of amino acid for each of the individual portions, such as by usingamino acid identification technique 1106. In some embodiments, each ofthe individual portions comprises a pulse pattern, and analyzing thedata further comprises determining a type of amino acid for one or moreof the portions based on its respective pulse pattern. In someembodiments, determining the type of amino acid further comprisesidentifying an amount of time within a portion when the data is above athreshold value and comparing the amount of time to a duration of timefor the portion. In some embodiments, determining the type of amino acidfurther comprises identifying at least one pulse duration for each ofthe one or more portions. In some embodiments, determining the type ofamino acid further comprises identifying at least one interpulseduration for each of the one or more portions.

Next, process 1300 proceeds to act 1306, where an amino acid sequencerepresentative of the polypeptide is outputted, such as via a userinterface. In some embodiments, the amino acid sequence includes aseries of amino acids corresponding to the portions.

An illustrative implementation of a computer system 1400 that may beused in connection with any of the embodiments of the technologydescribed herein is shown in FIG. 14. The computer system 1400 includesone or more processors 1410 and one or more articles of manufacture thatcomprise non-transitory computer-readable storage media (e.g., memory1420 and one or more non-volatile storage media 1430). The processor1410 may control writing data to and reading data from the memory 1420and the non-volatile storage device 1430 in any suitable manner, as theaspects of the technology described herein are not limited in thisrespect. To perform any of the functionality described herein, theprocessor 1410 may execute one or more processor-executable instructionsstored in one or more non-transitory computer-readable storage media(e.g., the memory 1420), which may serve as non-transitorycomputer-readable storage media storing processor-executableinstructions for execution by the processor 1410.

Computing device 1400 may also include a network input/output (I/O)interface 1440 via which the computing device may communicate with othercomputing devices (e.g., over a network), and may also include one ormore user I/O interfaces 1450, via which the computing device mayprovide output to and receive input from a user. The user I/O interfacesmay include devices such as a keyboard, a mouse, a microphone, a displaydevice (e.g., a monitor or touch screen), speakers, a camera, and/orvarious other types of I/O devices.

The above-described embodiments can be implemented in any of numerousways. For example, the embodiments may be implemented using hardware,software or a combination thereof. When implemented in software, thesoftware code can be executed on any suitable processor (e.g., amicroprocessor) or collection of processors, whether provided in asingle computing device or distributed among multiple computing devices.It should be appreciated that any component or collection of componentsthat perform the functions described above can be generically consideredas one or more controllers that control the above-discussed functions.The one or more controllers can be implemented in numerous ways, such aswith dedicated hardware, or with general purpose hardware (e.g., one ormore processors) that is programmed using microcode or software toperform the functions recited above.

In this respect, it should be appreciated that one implementation of theembodiments described herein comprises at least one computer-readablestorage medium (e.g., RAM, ROM, EEPROM, flash memory or other memorytechnology, CD-ROM, digital versatile disks (DVD) or other optical diskstorage, magnetic cassettes, magnetic tape, magnetic disk storage orother magnetic storage devices, or other tangible, non-transitorycomputer-readable storage medium) encoded with a computer program (i.e.,a plurality of executable instructions) that, when executed on one ormore processors, performs the above-discussed functions of one or moreembodiments. The computer-readable medium may be transportable such thatthe program stored thereon can be loaded onto any computing device toimplement aspects of the techniques discussed herein. In addition, itshould be appreciated that the reference to a computer program which,when executed, performs any of the above-discussed functions, is notlimited to an application program running on a host computer. Rather,the terms computer program and software are used herein in a genericsense to reference any type of computer code (e.g., applicationsoftware, firmware, microcode, or any other form of computerinstruction) that can be employed to program one or more processors toimplement aspects of the techniques discussed herein.

EXAMPLES Example 1. Edman Degradation by Chemical Cleavage

A surface-attached oligopeptide of approximately 3 to approximately 30amino acids (n=3-30) is provided, where amino acid residues R₁-R₃ can beany of the common 20 amino acids or an endogenously modified amino acid(e.g., modified by a post-translational modification). In theisothiocyanate N-terminal reaction, Step 1, an isothiocyanate X-NCS isadded to a vessel containing the surface-attached oligopeptide, where Xis phenyl (Ph), 4-NO₂Ph, 4-SO₃Ph, napthyl, benzyl, alkyl, or aderivative thereof. Step 1 is carried out under the following Conditionsto afford the X-NCS derivatized N-terminal amino acid: aqueous buffer pH4-10, MeOH or EtOH or IPA alcoholic co-solvents, trialkylamines inorganic solvents (DCM, THF, MeCN, DMF, and the like), 20° C. to 50° C.In the thiourea cleavage reaction, Step 2, an Acid or a Base is added toa vessel containing the X-NCS derivatized N-terminal amino acid, wherethe Acid is acetic acid, formic acid, trichloroacetic acid,trifluoroacetic acid, phosphoric acid, or hydrochloric acid, as neat oraqueous solutions, or where the Base is a trialkylamine or a bufferedtrialkylamine (e.g., Et₃NH⁺AcO⁻). Step 2 is carried out under thefollowing Conditions to afford the n−1 oligopeptide and thiohydantoinbyproduct: neat acid or with aqueous/organic co-solvents of any ratio,20° C. to 50° C.

Example 2. Solubilizing Linkers for Peptide Surface Immobilization

Seeking to improve oligopeptide solubility in aqueous buffer, it wasdetermined that peptide fragments could be conjugated witholigonucleotide linkers to both improve aqueous solubility and provide afunctional moiety for surface immobilization of peptides at the singlemolecule level. Different peptide-linker conjugates were synthesized,with example structures depicted in FIG. 15A for a peptide-DNA conjugateand a peptide-PEG conjugate. Linker conjugation was observed to greatlyenhance peptide solubility in aqueous solution for each of the differentpeptide-linker conjugates evaluated.

The peptide-linker conjugates were evaluated for amino acid cleavage atpeptide N-termini by N-terminal aminopeptidases (Table 6, below).

TABLE 6 Terminal amino acid cleavage of peptide-linker conjugates.SEQ ID Cleaved by Rat Cleaved by Entry Peptide NO. Class Linker APN PIP1 KF 70 positive oligo No 2 KKMKKM{LYS(N3)} 71 positive oligo No 3KKMKKM{LYS(N3)} 71 positive oligo-PEG No 4 KKMKKM{LYS(N3)} 71 positivePEG4 Yes 5 DDMDDM{LYS(N3)} 72 negative oligo Yes 6 FFMFFM{LYS(N3)} 73aromatic oligo Yes 7 AAMAAM{LYS(N3)} 74 hydrophobic oligo Yes 8FPFPFP{LYS(N3)} 75 aromatic oligo Yes 9 DPDPDP{LYS(N3)} 76 negativeoligo Yes 10 KPKPKP{LYS(N3)} 77 positive oligo No 11 KPKPKP{LYS(N3)} 77positive PEG4 Yes

The peptide-linker conjugates shown in Table 6 were incubated witheither proline iminopeptidase (“PIP”) or rat aminopeptidase N (“RatAPN”), and peptide cleavage was monitored by LCMS. An example of an LCMSdemonstrating cleavage of Entry 5 from Table 6 is shown in FIG. 15B. Allother cleavage reactions were measured in a similar manner. As shown inTable 6, while positively charged peptide-DNA conjugates (“oligo” and“oligo-PEG” linkers) were not cleaved by the aminopeptidases tested, allother conjugate classes (negatively charged, aromatic, hydrophobic) withDNA oligonucleotide linkers were cleaved. By comparison, the positivelycharged peptide-PEG conjugates were shown to be cleaved by at least oneof the aminopeptidases.

Using labeled peptide-linker conjugates, it was shown that peptides ofdifferent compositions could be immobilized to individual sample wellsurfaces for single molecule analysis. For these experiments, the DNAlinker was labeled with a dye (e.g., as depicted in FIG. 15A for thepeptide-DNA conjugate), and loading of different peptide-DNA conjugatesinto individual sample wells was measured by dye fluorescence. Anexample loading experiment is shown in FIG. 15C. By measuringfluorescence emission of a labeled peptide-DNA conjugate (50 pM), it wasdetermined that at least 18% of sample wells on a chip were loaded atsingle occupancy per sample well with a surface-immobilized conjugate.These experiments demonstrated that peptide-linker conjugates displayenhanced aqueous solubility compared to non-conjugated peptidecounterpart, that conjugated linkers do not prevent terminal amino acidcleavage of peptides by different aminopeptidases, and thatpeptide-linker conjugates of different compositions can be immobilizedto chip surfaces at the single molecule level.

Example 3. Exopeptidase Cleavage of Polypeptide Substrates

The cleavage capabilities of various aminopeptidases were tested. Theconditions and results for a set of cleavage assay experiments are shownin Table 7, including concentration of peptide substrate, concentrationof enzyme, buffer conditions, temperature, and incubation time. Cleavageof peptide substrates by the indicated enzymes was assayed using HighPerformance Liquid Chromatography (HPLC). The “HPLC assay cony” value inTable 7 indicates the percentage of the peptide substrate that wasconverted to cleavage product. To determine the “HPLC assay cony” value,two solutions were prepared containing the same starting concentrationof peptide. One solution was subjected to enzymatic digestion, while theother solution did not contain any enzyme, but was diluted with anequivalent amount of buffer used to store the enzyme. The reactions werequenched at the time indicated. The amount of reactant converted toproduct was determined by dividing the area of the peak obtained by HPLCof the starting material remaining after enzymatic digestion by the peakarea of the control solution of undigested peptide, and then multiplyingthis ratio by 100. In Table 7, “NH2” indicates an amine group, “yPIP”refers to Y. pestis proline iminopeptidase, “NPEPPS” refers topuromycin-sensitive aminopeptidase, “VPr” refers to Vibrio proteolyticusaminopeptidase, and “EDAPN” refers to L. pneumophila M1 aminopeptidase.

TABLE 7 Cleavage of peptide substrates by aminopeptidases. HPLC AssayEnzyme Peptide Substrate Conditions Temp/Time Conv Pdt yPIP GlyProArgPro5 mM peptide, 50 nM 30° C./1 hr 100% ProArgPro (SEQ ID NO: 84)enzyme, 10 mM MgCl₂, 10 mM Tris, 0.02% Tween-20 pH 8.0 yPIPDDPDDP{LYSN3}NH2 1 mM peptide, 700 nM 30° C./6 hrs 0% n/a(SEQ ID NO: 85) enzyme, 10 mM MgCl₂, 10 mM Tris, 0.02% Tween-20 pH 8.0yPIP AAMAAM{LYSN3}N 1 mM peptide, 700 nM 30° C./6 hrs 0% n/aH2 (SEQ ID NO: 74) enzyme, 10 mM MgCl₂, 10 mM Tris, 0.02% Tween-20pH 8.0 yPIP YPYPYP{LYSN3}NH2 600 mM peptide, 7 mM 30° C./1 hr 100%PYPYP{LYSN3} (SEQ ID NO: 86) enzyme, 10 mM MgCl₂, 10 NH2mM Tris, 0.02% Tween-20 (SEQ ID NO: 87) pH 8.0 yPIP FPFPFP{LYSN3}NH2600 mM peptide, 7 mM 30° C./1 hr 100% PFPFP{LYSN3}N (SEQ ID NO: 75)enzyme, 10 mM MgCl₂, 10 H2 mM Tris, 0.02% Tween-20 (SEQ ID NO: 88)pH 8.0 NPEPPS LeuTyr 5 mM 700 nM enzyme, 25 mM 37° C./1 hr 5% TyrHEPES, 1 mM Mg(OAc)₂, 1 mM DTT, pH 7.5 (Cy3B)n- FPFPFP{LYSN3}NH21 mM peptide, 14 mM 30° C./15 100% PFPFP{LYSN3}N yPIP (SEQ ID NO: 75)enzyme, 10 mM MgCl₂, 10 min H2 mM Tris, 0.02% Tween-20 (SEQ ID NO: 88)pH 8.0 (Cy3B)n- FPFPFP{LYSN3}NH2 1 mM peptide, 14 mM 30° C./15 100%PFPFP{LYSN3}N yPIP (SEQ ID NO: 75) enzyme, 200 mM Bis-tris min H2Propane, 30 mM KOAc, 25 (SEQ ID NO: 88) mM Mg(OAc)₂, 32 mM 3,4Dihydroxy-benzoic acid and 12 mM Nitrobenzoic acid P. MetTyr1 mM peptide, 1 mM 30° C./1 hr 7% Tyr falciparum enzyme, 2.5 mM ZnC1₂ 25M1 mM Tris pH 8.0 (Atto647 FPFPFP{LYSN3}NH2 1 mM peptide, 14 mM30° C./1 hr 100% PFPFP{LYSN3}N N)n-yPIP (SEQ ID NO: 75)enzyme, 10 mM MgCl₂, 10 H2 mM Tris, 0.02% Tween-20 (SEQ ID NO: 88)pH 8.0 Rat APN KKMKKMLys-Triazole- 1 mM peptide, 150 nM 30° C./1 hr >90%PEG4 Biotin enzyme, 2.5 mM ZnC1₂ 25 (SEQ ID NO: 89) mM Tris pH 8.0 yPIPFPFPFP{LYSN3}NH2 1 mM peptide, 7 mM 30° C./1 hr 100% PFPFP{LYSN3}N(SEQ ID NO: 75) enzyme, 10 mM MgCl₂, 10 H2 mM Tris, 0.02% Tween-20(SEQ ID NO: 88) pH 8.0 yPIP BCN PEG23 Biotin 1 mM peptide, 7 mM30° C./1 hr 100% PFPFP{LYS}BCN conjugate enzyme, 10 mM MgCl₂, 10 etcmM Tris, 0.02% Tween-20 (SEQ ID NO: 88) pH 8.0 P. DDMDDM{LYSN3}N1 mM peptide, 1 mM 70° C./1 hr 60% horikoshii H2 (SEQ ID NO: 72)enzyme, 2.5 mM ZnCl₂, 25 Tet mM Tris pH 8.0 GluAsp- DDMDDM{LYSN3}N1 mM peptide, 1 mM 30° C./1 hr 100% APN H2 (SEQ ID NO: 72)enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 P. AAPAAP{LYSN3}NH21 mM peptide, 1 mM 70° C./1 hr 100% APAAP{LYSN3} horikoshii(SEQ ID NO: 90) enzyme, 2.5 mM ZnCl₂, 25 NH2 Tet mM Tris pH 8.0(SEQ ID NO: 91) P. YYPYYP{LYSN3}NH2 1 mM peptide, 1 mM 70° C./1 hr 100%YPYYP{LYSN3} horikoshii (SEQ ID NO: 92) enzyme, 2.5 mM ZnCl₂, 25 NH2 TetmM Tris pH 8.0 (SEQ ID NO: 93) P. FFPFFP{LYSN3}NH2 1 mM peptide, 1 mM70° C./1 hr 100% FPFFP{LYSN3}N horikoshii (SEQ ID NO: 94)enzyme, 2.5 mM ZnCl₂, 25 H2 Tet mM Tris pH 8.0 (SEQ ID NO: 95) Rat APNRRPRRP{LYSN3}NH2 1 mM peptide, 50 nM 30° C./1 hr 55% RPRRP{LYSN3}(SEQ ID NO: 96) enzyme, 2.5 mM ZnCl₂, 25 NH2 mM Tris pH 8.0(SEQ ID NO: 97) Rat APN AAPAAP{LYSN3}NH2 1 mM peptide, 50 nM 30° C./1 hr100% APAAP{LYSN3} (SEQ ID NO: 98) enzyme, 2.5 mM ZnCl₂, 25 NH2mM Tris pH 8.0 (SEQ ID NO: 99) Rat APN KKPKKP{LYSN3}NH21 mM peptide, 50 nM 30° C./1 hr 85% KPKKP{LYSN3} (SEQ ID NO: 100)enzyme, 2.5 mM ZnCl₂, 25 NH2 mM Tris pH 8.0 (SEQ ID NO: 101) Rat APNKKMKKM{LYSN3}N 1 mM peptide, 50 nM 30° C./1 hr 50% KMKKM{LYSN3H2 (SEQ ID NO: 71) enzyme, 2.5 mM ZnCl₂, 25 }NH2 mM Tris pH 8.0(SEQ ID NO: 102) VPr RRPRRP{LYSN3}NH2 1 mM peptide, 2 mM 30° C./1 hr100% (SEQ ID NO: 96) enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 VPrAAPAAP{LYSN3}NH2 1 mM peptide, 2 mM 30° C./1 hr 100% APAAP{LYSN3}(SEQ ID NO: 98) enzyme, 2.5 mM ZnCl₂, 25 NH2 mM Tris pH 8.0(SEQ ID NO: 99) VPr KKPKKP{LYSN3}NH2 1 mM peptide, 2 mM 30° C./1 hr 100%KPKKP{LYSN3} (SEQ ID NO: 100) enzyme, 2.5 mM ZnC1₂ 25 NH2 mM Tris pH 8.0(SEQ ID NO: 101) VPr YYPYYP{LYSN3}NH2 1 mM peptide, 2 mM 30° C./1 hr 50%YPYYP{LYSN3} (SEQ ID NO: 92) enzyme, 2.5 mM ZnCl₂, 25 NH2 mM Tris pH 8.0(SEQ ID NO: 93) VPr FFPFFP{LYSN3}NH2 1 mM peptide, 2 mM 30° C./1 hr 100%FPFFP{LYSN3}N (SEQ ID NO: 94) enzyme, 2.5 mM ZnCl₂, 25 H2 mM Tris pH 8.0(SEQ ID NO: 95) VPr AAMAAM{LYSN3}N 1 mM peptide, 2 mM 30° C./1 hr 100%multiple pdts H2 (SEQ ID NO: 74) enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0VPr KKMKKM{LYSN3}N 1 mM peptide, 2 mM 30° C./1 hr 100% multiple pdtsH2 (SEQ ID NO: 89) enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 VPrYYMYYM{LYSN3}N 1 mM peptide, 2 mM 30° C./1 hr >90% multiple pdtsH2 (SEQ ID NO: 103) enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 yPIPAttor6g-1kPEG- 44 mM Peptide, 7 mM 30° C./0.5 <10% PAAAFK-1kPEG-DPAAAFK{LysN3}- enzyme, 200 mM Bis-tris hr Biotin 1kPEG-BiotinPropane, 30 mM KOAc, 25 (SEQ ID NO: 105) (SEQ ID NO: 104)mM Mg(OAc)₂, 32 mM 3,4 Dihydroxy-benzoic acid and12 mM Nitrobenzoic acid + PCD + TXV PfuPIP FPFPFP{LYSN3}NH21 mM Peptide, 1 μM enzyme, 80° C./0.5 100% PFPFP{LYSN3}N (SEQ ID NO: 75)1 mM CoCl₂, 50 mM hr H2 HEPES, 50 mM KC1 (SEQ ID NO: 88) PfuPIPAttor6g-1kPEG- 1 mM Peptide, 1 μM enzyme, 80° C./0.5 40% PAAAFK-1kPEG-DPAAAFK{LysN3}- 1 mM CoCl₂, 50 mM hr Biotin 1kPEG-BiotinHEPES, 50 mM KC1 (SEQ ID NO: 105) (SEQ ID NO: 104) yPIPAttor6g-1kPEG-ODN- 20 μM Q24 conjugate, 30 μM 30° C./0.3 100%PAAAFK-1kPEG- DD60-Biotin ypip, lx Mg buffer hr Biotin (SEQ ID NO: 105)yPIP FPFPFP{LYSN3}NH2 1 mM Peptide, 7 μM 37° C./0.5 100% PFPFP{LYSN3}N(SEQ ID NO: 75) Enzyme, 50 mM MOPS, 10 hr H2 mM Mg(OAc)₂ pH 8.0(SEQ ID NO: 88) yPIP Attor6g-1kPEG-ODN- 10 μM Peptide, 7 μM 37° C./0.5100% PAAAFK-1kPEG- DD60-Biotin Enzyme, 50 mM MOPS, 10 hr BiotinmM Mg(OAc)₂ pH 8.0 (SEQ ID NO: 105) PfuPIP FPFPFP{LYSN3}NH21 mM Peptide, 1 μM 80° C./0.5 40% PFPFP{LYSN3}N (SEQ ID NO: 75)Enzyme, 50 mM MOPS, 10 hr H2 mM Mg(OAc)₂ pH 8.0 (SEQ ID NO: 88) yPIP-FPFPFP{LYSN3}NH2 1 mM Peptide, 2.1 μM 37° C./0.5 100% PFPFP{LYSN3}N Q24-(SEQ ID NO: 75) Enzyme, 50 mM MOPS, 10 hr H2 Cy3B mM Mg(OAc)₂ pH 8.0(SEQ ID NO: 88) yPIP- YPYPYP{LYSN3}NH2 1 mM Peptide, 100 nM 37° C./0.5100% YPYYP {LYSN3} Q24- (SEQ ID NO: 86) Enzyme, 1X CB2 hr NH2 Rho6G(SEQ ID NO: 93) yPIP- YPYPYP{LYSN3}NH2 1 mM Peptide, ~5 μM 37° C./0.5<15% YPYYP{LYSN3} Q24Dark (SEQ ID NO: 86) Enzyme, 1X CB2 hr NH2(SEQ ID NO: 93) Rat APN QP5-Atto649N 37 μM Peptide, 100 nM37° C./0.5 >95% (KAAAAAAFK{LYSN Enzyme, 2.5 mM ZnCl₂, 25 hr. 3}NH2)mM Tris pH 8.0 (SEQ ID NO: 106) VPr QP5-Atto649N 37 μM Peptide, 8 μM37° C./0.5 100% (KAAAAAAFK{LYSN Enzyme, 2.5 mM ZnCl₂, 25 hr 3}NH2)mM Tris pH 8.0 (SEQ ID NO: 106) K287pA YPYPYP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr 100% PYPYPK zF-Cy3 (SEQ ID NO: 86)Enzyme, 50 mM MOPS, 10 (SEQ ID NO: 83) yPIP mM Mg(OAc)₂ pH 8.0 V.AAPAAP{LYSN3}NH2 1 mM Peptide, 1 μM 37° C./1 hr ~05% cholera(SEQ ID NO: 98) Enzyme, 2.5 mM ZnCl₂, 25 APT mM Tris pH 8.0 Bst M28AAPAAP{LYSN3}NH2 1 mM Peptide, 1 μM 37° C./1 hr 100% (SEQ ID NO: 98)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 Taq APT AAPAAP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr >90% (SEQ ID NO: 98)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 V. YYPYYP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr 5% cholera (SEQ ID NO: 92)Enzyme, 2.5 mM ZnCl₂, 25 APT mM Tris pH 8.0 Bst M28 YYPYYP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr 10% (SEQ ID NO: 92)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 Taq APT YYPYYP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr 30% (SEQ ID NO: 92)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 V. FFPFFP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr >95% cholera (SEQ ID NO: 94)Enzyme, 2.5 mM ZnCl₂, 25 APT mM Tris pH 8.0 Bst M28 FFPFFP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr 30% (SEQ ID NO: 94)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 Taq APT FFPFFP{LYSN3}NH21 mM Peptide, 1 μM 37° C./1 hr 60% (SEQ ID NO: 94)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 V. YYMYYM{LYSN3}N1 mM Peptide, 1 μM 37° C./1 hr 30% cholera H2 (SEQ ID NO: 103)Enzyme, 2.5 mM ZnCl₂, 25 APT mM Tris pH 8.0 Bst M28 YYMYYM{LYSN3}N1 mM Peptide, 1 μM 37° C./1 hr >50% multiple (N-1, H2 (SEQ ID NO: 103)Enzyme, 2.5 mM ZnCl₂, 25 -2, -3, etc.) mM Tris pH 8.0 Taq APTYYMYYM{LYSN3}N 1 mM Peptide, 1 μM 37° C./1 hr 85% multiple (N-1,H2 (SEQ ID NO: 103) Enzyme, 2.5 mM ZnCl₂, 25 -2, -3, etc.)mM Tris pH 8.0 Cy3B- YPYPYP{LYSN3}NH2 1 mM Peptide, 7 μM 37° C./0.5 hr100% Q24- (SEQ ID NO: 86) Enzyme, 10 mM MgCl₂, 50 pAzF- mM MOPS yPIPCy3B- FFPFFP{LYSN3}NH2 1 mM Peptide, 10 μM 37° C./0.5 hr 50% BstTET(SEQ ID NO: 94) Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 Cy3B-FFPFFP{LYSN3}NH2 1 mM Peptide, 20 μM 37° C./0.5 hr 100% taqAPT(SEQ ID NO: 94) Enzyme, 2.5 mM ZnCl₂, 25 2nd peak mM Tris pH 8.0 Cy3B-FFPFFP{LYSN3}NH2 1 mM Peptide, 20 μM 37° C./0.5 hr 100% taqAPT(SEQ ID NO: 94) Enzyme, 2.5 mM ZnCl₂, 25 4th peak mM Tris pH 8.0 PhaloM2RRPRRP{LYSN3}NH2 1 mM Peptide, 1 μM 37° C./0.5 hr 30% multiple, even 8(SEQ ID NO: 96) Enzyme, 2.5 mM ZnCl₂, 25 higher mass mM Tris pH 8.0 yPAPRRPRRP{LYSN3}NH2 1 mM Peptide, 1 μM 37° C./0.5 hr 100% (SEQ ID NO: 96)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 yPAP AAPAAP{LYSN3}NH21 mM Peptide, 1 μM 37° C./0.5 hr 100% (SEQ ID NO: 98)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 yPAP KKPKKP{LYSN3}NH21 mM Peptide, 1 μM 37° C./0.5 hr 100% (SEQ ID NO: 100)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 yPAP YYPYYP{LYSN3}NH21 mM Peptide, 1 μM 37° C./0.5 hr 100% (SEQ ID NO: 92)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 PhaloM2 FFPFFP{LYSN3}NH21 mM Peptide, 1 μM 37° C./0.5 hr >80% 8 (SEQ ID NO: 94)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 yPAP FFPFFP{LYSN3}NH21 mM Peptide, 1 μM 37° C./0.5 hr >80% (SEQ ID NO: 94)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 V. QP5-Atto649N1 mM Peptide, 5 μM 37° C./0.5 hr 100% cholera (KAAAAAAFK{LYSNEnzyme, 2.5 mM ZnCl₂, 25 APT 3}NH2) mM Tris pH 8.0 (SEQ ID NO: 106) yPAPYYPYYP{LYSN3}NH2 1 mM Peptide, 2 μM 37° C./0.5 hr  10% (SEQ ID NO: 92)Enzyme, 2.5 mM ZnCl₂, 25 mM Tris pH 8.0 V. RRPRRP{LYSN3}NH21 mM peptide, 2 μM enzyme, 30° C./1 hr >90% anguillarum (SEQ ID NO: 96)2.5 mM ZnCl₂, 25 mM Tris APN pH 8.0 V. AAPAAP{LYSN3}NH21 mM peptide, 2 μM enzyme, 30° C./1 hr 50% anguillarum (SEQ ID NO: 98)2.5 mM ZnCl₂, 25 mM Tris APN pH 8.0 V. KKPKKP{LYSN3}NH21 mM peptide, 2 μM enzyme, 30° C./1 hr <5% anguillarum (SEQ ID NO: 100)2.5 mM ZnCl₂, 25 mM Tris APN pH 8.0 VPr FYPLPWPDDDY{LYS300 μM Peptide, 4 μM 37° C./0.5 100% N3}NH2 enzyme, 10 mM MgCl₂, 50 hr(SEQ ID NO: 107) mM MOPS pH 8.0 yPIP YPLPWPDDDY{LYSN300 μM Peptide, 7 μM 37° C./0.5 100% 3}NH2 enzyme, 10 mM MgCl₂, 50 hr(SEQ ID NO: 108) mM MOPS pH 8.0 VPr PLPWPDDDY{LYSN3 300 μM Peptide, 4 μM37° C./0.5 100% }NH2 enzyme, 10 mM MgCl₂, 50 hr (SEQ ID NO: 109)mM MOPS pH 8.0 yPIP LPWPDDDY{LYSN3} 300 μM Peptide, 7 μM 37° C./0.5 100%NH2 (SEQ ID NO: 110)  enzyme, 10 mM MgCl₂, 50 hr mM MOPS pH 8.0 hTETFYPLPWPDDDY{LYS 200 μM Peptide, 2 μM 37° C./1 hr 55% YPLPWPDDDY{ N3}NH2enzyme, 2.5 mM ZnCl₂, 25 LYSN3}NH2 (SEQ ID NO: 107) mM Tris pH 8.0(SEQ ID NO: 108) hTET FYPLPWPDDDY{LYS 200 μM Peptide, 2 μM 37° C./1 hr55% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM MgCl₂, 50 LYSN3}NH2(SEQ ID NO: 107) mM MOPS pH 8.0 (SEQ ID NO: 108) Pro VPrAFYPLPWPDDDY{LYS 200 μM Peptide, 2.2 μM 37° C./1 hr 6% YPLPWPDDDY{ mbrN3}NH2 enzyme, 10 mM MgCl₂, 50 LYSN3}NH2 (SEQ ID NO: 107) mM MOPS pH 8.0(SEQ ID NO: 108) ThrCut- FYPLPWPDDDY{LYS 200 μM Peptide, 2.1 μM37° C./1 hr 40% YPLPWPDDDY{ ProVPrA N3}NH2 enzyme, 10 mM MgCl₂, 50LYSN3}NH2 mbr (SEQ ID NO: 107) mM MOPS pH 8.0 (SEQ ID NO: 108) VPrFYPLPWPDDDY{LYS 200 μM Peptide, 4 μM 37° C./0.5 100% YPLPWPDDDY{ N3}NH2enzyme, 10 mM MgCl₂, 50 hr LYSN3}NH2 (SEQ ID NO: 107) mM MOPS pH 8.0(SEQ ID NO: 108) ProVPrA FYPLPWPDDDY{LYS 200 μM Peptide, 4 μM 37° C./0.550% YPLPWPDDDY{ mbr N3}NH2 enzyme, 10 mM MgCl₂, 50 hr LYSN3}NH2(SEQ ID NO: 107) mM MOPS pH 8.0 (SEQ ID NO: 108) ThrCut- FYPLPWPDDDY{LYS200 μM Peptide, 4 μM 37° C./0.5 40% YPLPWPDDDY{ ProVPrA N3}NH2enzyme, 10 mM MgCl₂, 50 hr LYSN3}NH2 mbr (SEQ ID NO: 107) mM MOPS pH 8.0(SEQ ID NO: 108) VPr FWPLPWPDDDY{LYS 200 μM Peptide, 4 μM 37° C./1 hr100% N3}NH2 enzyme, 10 mM MgCl₂, 50 (SEQ ID NO: 107) mM MOPS pH 8.0 VPrFWPLPWPDDDY{LYS 200 μM Peptide, 4 μM 37° C./1 hr 96% pAzF N3}NH2enzyme, 10 mM MgCl₂, 50 (SEQ ID NO: 107) mM MOPS pH 8.0 ThrCut-FWPLPWPDDDY{LYS 200 μM Peptide, 6.4 μM 37° C./1 hr 100% Pro VPrA N3}NH2enzyme, 10 mM MgCl₂, 50 mbr Cy3 (SEQ ID NO: 107) mM MOPS pH 8.0 clickedhTET PLPWPDDDY{LYSN3 200 μM Peptide, 4 μM 37° C./1 hr 100% }NH2enzyme, 10 mM MgCl₂, 50 (SEQ ID NO: 109) mM MOPS pH 8.0 VPrFWPLPWPDDDY{LYS 400 μM Peptide, 8 μM 37° C./1 hr 100% N3}NH2enzyme, 50 mM HEPESpH (SEQ ID NO: 107) 8.0, 300 mM NaCl, 1 mMDTT, 5% Glycerol, 32 mM PCA VPr FYPLPWPDDDY{LYS 200 μM Peptide, 8 μMRT/1 hr 100% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM MgCl₂, 50 LYSN3}NH2(SEQ ID NO: 107) mM MOPS pH 5.0 (SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS200 μM Peptide, 8 μM RT/0.5 hr 100% YPLPWPDDDY{ N3}NH2enzyme, 10 mM MgCl₂, 50 LYSN3}NH2 (SEQ ID NO: 107) mM MOPS pH 5.0(SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 200 μM Peptide, 8 μM RT/1 hr 100%YPLPWPDDDY{ N3}NH2 enzyme, 10 mM MgCl₂, 50 LYSN3}NH2 (SEQ ID NO: 107)mM MOPS pH 5.0 (SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 200 μM Peptide, 8 μMRT/0.5 hr 100% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM MgCl₂, 50 LYSN3}NH2(SEQ ID NO: 107) mM MOPS pH 5.0 (SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS600 μM Peptide, 0.8 μM RT/0.5 hr 100% YPLPWPDDDY{ N3}NH2enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 (SEQ ID NO: 107) 50 mM MOPS pH 5.5(SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 600 μM Peptide, 0.8 μM RT/0.5 hr100% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2(SEQ ID NO: 107) 50 mM MOPS pH 6.5 (SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS600 μM Peptide, 0.8 μM RT/0.5 hr 100% YPLPWPDDDY{ N3}NH2enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 (SEQ ID NO: 107) 50 mM MOPS pH 7.5(SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 600 μM Peptide, 0.8 μM RT/0.5 hr100% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2(SEQ ID NO: 107) 50 mM MOPS pH 8.5 (SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS1200 μM Peptide, 0.08 μM RT/0.5 hr 50% YPLPWPDDDY{ N3}NH2enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 (SEQ ID NO: 107) 50 mM MOPS pH 5.5(SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 1200 μM Peptide, 0.08 μM RT/0.5 hr100% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2(SEQ ID NO: 107) 50 mM MOPS pH 6.5 (SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS1200 μM Peptide, 0.08 μM RT/0.5 hr 100% YPLPWPDDDY{ N3}NH2enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 (SEQ ID NO: 107) 50 mM MOPS pH 7.5(SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 1200 μM Peptide, 0.08 μM RT/0.5 hr100% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 (SEQ ID NO: X)50 mM MOPS pH 8.5 (SEQ ID NO: 108) VPr QP15 1200 μM Peptide, 0.008 μMRT/0.5 hr 1.40% YPLPWPDDDY{ FYPLPWPDDDY{LYS enzyme, 10 mM Mg(OAc)₂,LYSN3}NH2 N3}NH2 50 mM MOPS pH 5.5 (SEQ ID NO: 108) (SEQ ID NO: 107) VPrQP15 1200 μM Peptide, 0.008 μM RT/0.5 hr 56% YPLPWPDDDY{ FYPLPWPDDDY{LYSenzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 N3}NH2 50 mM MOPS pH 6.5(SEQ ID NO: 108) (SEQ ID NO: 107) VPr FYPLPWPDDDY{LYS1200 μM Peptide, 0.008 μM RT/0.5 hr 100% YPLPWPDDDY{ N3}NH2enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 (SEQ ID NO: 107) 50 mM MOPS pH 7.5(SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 1200 μM Peptide, 0.008 μM RT/0.5 hr100% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2(SEQ ID NO: 107) 50 mM MOPS pH 8.5 (SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS1200 μM Peptide, 800 pM RT/0.5 hr 2.70% YPLPWPDDDY{ N3}NH2enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2 (SEQ ID NO: 107) 50 mM MOPS pH 7.5(SEQ ID NO: 108) VPr FYPLPWPDDDY{LYS 1200 μM Peptide, 800 pM RT/0.5 hr6.80% YPLPWPDDDY{ N3}NH2 enzyme, 10 mM Mg(OAc)₂, LYSN3}NH2(SEQ ID NO: 107) 50 mM MOPS pH 8.5 (SEQ ID NO: 108) VPr FAAAWPDDDF1600 μM Peptide, 8 μM RT/0.5 hr 100% WPDDF1 (SEQ ID NO: 11)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 112 50 mM MOPS pH 8 VPr WAAAFPDDDF1600 μM Peptide, 8 μM RT/0.5 hr 100% FPDDF1 (SEQ ID NO: 13)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 114) 50 mM MOPS pH 8 VPr WAAAFPDDDF1300 μM Peptide, 8 μM RT/0.5 hr 100% FPDDF1 (SEQ ID NO:13)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 114) 50 mM MOPS pH 8 VPr WAAAFPDDDF1300 μM Peptide, 8 μM RT/0.5 hr 100% FPDDF1 (SEQ ID NO: 13)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 114) 50 mM MOPS pH 8, 0.2% Tween20VPr WAAAFPDDDF1 300 μM Peptide, 8 μM RT/0.5 hr 30% 70% -3, 30% -4(SEQ ID NO: 13) enzyme, 50/50 MOPSMg buffer/RB1 VPr WAAAFPDDDF1300 μM Peptide, 8 μM RT/0.5 hr 5% Almost all -1, -2 (SEQ ID NO: 13)enzyme, RB2 + Mg and -3 products VPr WAAAFPDDDF1 300 μM Peptide, 8 μMRT/0.5 hr 100% FPDDF1 (SEQ ID NO: 13) enzyme, RB4 (SEQ ID NO: 114) VPrWAAAFPDDDF1 300 μM Peptide, 8 μM RT/0.5 hr 70% 30% -3, 70% -4(SEQ ID NO: 13) enzyme, 10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc VPrWAAAFPDDDF1 300 μM Peptide, 8 μM RT/2 hr 100% FPDDF1 (SEQ ID NO: 13)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 114) 50 mM MOPS pH 8, 60 mM KOAc VPrWAAAFPDDDF1 300 μM Peptide, 8 μM 37° C./2 hr 100% FPDDF1 (SEQ ID NO: 13)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 114) 50 mM MOPS pH 8, 60 mM KOAc VPrFAAAYPDDDF1 600 μM Peptide, 8 μM RT/0.5 hr 100% YPDDF1 (SEQ ID NO: 11)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 115) 50 mM MOPS pH 8, 60 mM KOAc VPrFAAAYPDDDF1 600 μM Peptide, 0.8 μM RT/0.5 hr 50% YPDDF1 (SEQ ID NO: 11)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 115) 50 mM MOPS pH 8, 60 mM KOAc VPrFAAAYPDDDF1 600 μM Peptide, 0.08 μM RT/0.5 hr 5% YPDDF1 (SEQ ID NO: 11)enzyme, 10 mM Mg(OAc)₂, (SEQ ID NO: 115) 50 mM MOPS pH 8, 60 mM KOAc VPrRRPFQQ 1 mM Peptide, 1 μM enzyme, RT/0.5 hr 79% RPFQQ (SEQ ID NO: 116)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 117) MOPS pH 8, 60 mM KOAc VPr AAPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% APFQQ (SEQ ID NO: 118)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 119) MOPS pH 8, 60 mM KOAc VPr KKPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 32% KPFQQ (SEQ ID NO: 120)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 121) MOPS pH 8, 60 mM KOAc VPr YYPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 56% YPFQQ (SEQ ID NO: 122)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 123) MOPS pH 8, 60 mM KOAc VPr FFPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% FPFQQ (SEQ ID NO: 124)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 125) MOPS pH 8, 60 mM KOAc VPr DDPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% DPFQQ (SEQ ID NO: 126)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 127) MOPS pH 8,60 mM KOAc VPr EEPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% EPFQQ (SEQ ID NO: 128)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 129) MOPS pH 8, 60 mM KOAc VPr NNPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 15% NPFQQ (SEQ ID NO: 130)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 131) MOPS pH 8, 60 mM KOAc VPr QQPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 66% QPFQQ (SEQ ID NO: 132)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 133) MOPS pH 8, 60 mM KOAc VPr VVPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% VPFQQ (SEQ ID NO: 134)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 135) MOPS pH 8, 60 mM KOAc VPr IIPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% IPFQQ (SEQ ID NO: 136)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 137) MOPS pH 8, 60 mM KOAc VPr LLPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% LPFQQ (SEQ ID NO: 138)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 139) MOPS pH 8, 60 mM KOAc VPr SSPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 48% SPFQQ (SEQ ID NO: 140)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 141) MOPS pH 8, 60 mM KOAc VPr TTPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% TPFQQ (SEQ ID NO: 142)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 143) MOPS pH 8, 60 mM KOAc VPr CCPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 70% CPFQQ (SEQ ID NO: 144)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 145) MOPS pH 8, 60 mM KOAc VPr WWPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 82% WPFQQ (SEQ ID NO: 146)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 147) MOPS pH 8, 60 mM KOAc VPr MMPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% MPFQQ (SEQ ID NO: 148)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 149) MOPS pH 8, 60 mM KOAc VPr PPPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% PPFQQ (SEQ ID NO: 150)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 151) MOPS pH 8, 60 mM KOAc VPr GGPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 8% GPFQQ (SEQ ID NO: 152)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 153) MOPS pH 8, 60 mM KOAc VPr HHPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 12% HPFQQ (SEQ ID NO: 154)10 mM Mg(OAc)₂, 50 mM (SEQ ID NO: 155) MOPS pH 8, 60 mM KOAc yPIP RRPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 116)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP AAPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 118)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP KKPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 120)10 mM Mg(OAc)₂, 50 mM MOPS pH 8,60 mM KOAc yPIP YYPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 122)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP FFPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 124)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP DDPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 126)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP EEPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 128)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP NNPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 130)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP QQPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 132)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP VVPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 134)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP IIPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 136)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP LLPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 138)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP SSPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 140)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP TTPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 142)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP CCPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 144)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP WWPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 146)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP MMPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 148)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP PPPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 26% multiple pAzF (SEQ ID NO: 150)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP GGPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 152)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc yPIP HHPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% N/A pAzF (SEQ ID NO: 154)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET RRPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 116)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET AAPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 118)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET KKPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 120)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET YYPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 99% (SEQ ID NO: 122)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET FFPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 124)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET DDPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 4% (SEQ ID NO: 126)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET EEPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 128)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET NNPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 69% (SEQ ID NO: 130)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET QQPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 63% (SEQ ID NO: 132)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET VVPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 134)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET IIPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 136)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET LLPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 138)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET SSPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 140)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET TTPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 142)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET CCPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 32% (SEQ ID NO: 144)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET WWPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 4% (SEQ ID NO: 146)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET MMPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 148)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET PPPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 150)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET GGPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 33% (SEQ ID NO: 152)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc hTET HHPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 26% (SEQ ID NO: 154)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET RRPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 116)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET AAPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 118)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET KKPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 120)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET YYPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 122)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET FFPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 65% (SEQ ID NO: 124)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET DDPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 86% (SEQ ID NO: 126)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET EEPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 93% (SEQ ID NO: 128)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET NNPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 81% (SEQ ID NO: 130)10 mM Mg(OAc)₂, 50 mM MOPS pH 8,60 mM KOAc PfuTET QQPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 132)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET VVPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 134)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET IIPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 136)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET LLPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 99% (SEQ ID NO: 138)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET SSPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 90% (SEQ ID NO: 140)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET TTPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 142)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET CCPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 72% (SEQ ID NO: 144)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET WWPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 37% (SEQ ID NO: 146)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET MMPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 100% (SEQ ID NO: 148)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET PPPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 150)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET GGPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 0% (SEQ ID NO: 152)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc PfuTET HHPFQQ1 mM Peptide, 1 μM enzyme, RT/0.5 hr 19% (SEQ ID NO: 154)10 mM Mg(OAc)₂, 50 mM MOPS pH 8, 60 mM KOAc EDAPN RRPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 116)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN AAPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 118)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN KKPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 120)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN YYPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 122)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN FFPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 124)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN DDPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 21% (Glu/Asp (SEQ ID NO: 126)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN EEPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 31% (Glu/Asp (SEQ ID NO: 128)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN NNPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 130)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN QQPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 132)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN VVPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 134)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN IIPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 42% (Glu/Asp (SEQ ID NO: 136)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN LLPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 138)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN SSPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 140)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN TTPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 142)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN CCPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 144)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc EDAPN WWPFQQ1 mM Peptide, 1.3 μM RT/0.5 hr 0% (Glu/Asp (SEQ ID NO: 146)enzyme, 10 mM Mg(OAc)₂, APN) 50 mM MOPS pH 8, 60 mM KOAc PfuTETYAAFAAWADDDW1 1 mM Peptide, 1.0 μM RT/0.5 hr Distri- Mainly ADDDWK(SEQ ID NO: 156) enzyme, 10 mM Mg(OAc)₂, bution (SEQ ID NO: 157)50 mM MOPS pH 8, 60 mM KOAc hTET YAAFAAWADDDW1 1 mM Peptide, 1.0 μMRT/0.5 hr 100% WADDDWK (SEQ ID NO: 156) enzyme, 10 mM Mg(OAc)₂,(SEQ ID NO: 158) 50 mM MOPS pH 8, 60 mM KOAc VPr YAAFAAWADDDW11 mM Peptide, 1.2 μM RT/0.5 hr Distri- Mainly ADDDWK (SEQ ID NO: 156)enzyme, 10 mM Mg(OAc)₂, bution (SEQ ID NO: 157) 50 mM MOPS pH 8, 60 mMKOAc

A summary of amino acid cleavage activities for select exopeptidases ofTable 7 is shown in FIG. 16. Specific cleavage activities are shown forthe following enzymes: “cVPr” (V. proteolyticus aminopeptidase), “yPIP”(Y. pestis proline iminopeptidase), “D/E APN” (L. pneumophila M1Aminopeptidase), hTET (Pyrococcus horikoshii TET aminopeptidase), andPfu API (“PfuTET” in Table 7). Specific activities with respect toterminal amino acids are classified as shown, with single-letterabbreviations used for amino acids (“XP-” represents any terminal aminoacid (X) having an adjacent, or penultimate, proline (P) residue).

Example 4. Terminal Amino Acid Cleavage of Immobilized Peptides atSingle Molecule Level

Assays for on-chip amino acid cleavage of immobilized peptides weredeveloped using labeled peptide conjugates. The assays were designed toprovide a method for determining enzymatic recognition and cleavageactivity of exopeptidases toward immobilized peptides, which couldpermit measurement of kinetic binding parameters and general bindingaffinities.

To evaluate N-terminal amino acid cleavage of a peptide, a dye labeledpeptide was designed and synthesized which contained an N-terminalaspartate that was attached to the dye by way of a PEG spacer. Thispeptide also contained a proline residue adjacent to the modifiedaspartate that is recognized specifically by the enzyme prolineiminopeptidase (from Yersinia pestis, known elsewhere and referred toherein as “yPIP”). The enzyme yPIP should cleave only an N-terminalamino acid upstream from a proline residue.

After showing that this and other labeled peptides were efficiently cutby yPIP in bulk (e.g., as described in Example 2), an on-chipdye/peptide conjugate assay was developed to observe N-terminal aminoacid cleavage at the single molecule level. FIG. 17A shows a generalscheme for the dye/peptide conjugate assay (inset panel). As shown, apeptide having a label attached to an N-terminal amino acid via a spaceris immobilized to a surface by way of a linker. After being exposed topeptidase, N-terminal amino acid cleavage results in the removal of thelabeled residue from a detectable observation volume and is measured bya concomitant loss in signal from the label. The enzyme-peptide complexto the right of the inset panel generically depicts the N-terminalcleavage site.

FIG. 17A shows a labeled peptide construct (at bottom) that was designedand synthesized for use in the dye/peptide conjugate assay. In theseexperiments, a rhodamine dye (ATTO Rho6G) was attached to an N-terminalaspartate residue of a peptide having a penultimate proline residue atthe N-terminus. As shown, the peptide was further conjugated to asolubilizing DNA linker with a biotin moiety for surface immobilization.

The labeled peptide conjugate was loaded onto a glass chip having anarray of sample wells. Images of the chip were acquired before and afterloading to determine the percent loading of sample wells at singleoccupancy by rhodamine fluorescence. The enzyme yPIP was then introducedonto the loaded chip and allowed to incubate for two hours at 37° C. Animage of the chip following the introduction of yPIP was taken and thepercentage of green dyes lost were calculated to evaluate N-terminalamino acid cleavage. FIG. 17B shows imaging results from an experimentwhich displayed 6-7% loading in the loading stage and 91% loss of signalin previously loaded wells after incubation with yPIP, which wasindicative of N-terminal amino acid cleavage. FIG. 17C showsrepresentative signal traces from these experiments, which demonstrate adetected increase in dye signal upon loading of labeled peptide and adetected loss in dye signal following exposure to yPIP.

As further confirmation of N-terminal amino acid cleavage at the singlemolecule level, on-chip FRET assays were developed to evaluateexopeptidase recognition and cleavage activity. FIG. 18A genericallydepicts a FRET peptide conjugate assay (panel A) and a FRET enzymeconjugate assay (panel B). In the FRET peptide conjugate assay (panelA), an immobilized peptide construct includes a FRET donor labelattached to the linker and a FRET acceptor label attached at theN-terminus. N-terminal amino acid cleavage is detected by a loss insignal from the FRET acceptor label when exposed to peptidase.Additionally, this design permits monitoring loading of the peptideconjugate throughout an experiment by following emission from the FRETdonor label.

In the FRET enzyme conjugate assay (panel B), an immobilized peptideconstruct includes a first label of a FRET pair attached to the linkerand a peptidase is labeled with a second label of the FRET pair.N-terminal amino acid cleavage is detected by an enhancement influorescence attributable to FRET interactions, which would occur withsufficient proximity of peptidase to peptide and with sufficientresidence time at the N-terminus. Additionally, this assay permitsevaluating processive amino acid cleavage by a processive exopeptidaseby detecting an increasing FRET signal over time with processivecleavage.

FIG. 18A also shows a FRET peptide construct under panel A that wasdesigned and synthesized for use in the FRET peptide conjugate assay ofpanel A. As shown, the FRET peptide construct included a rhodamine dye(ATTO 647N) attached to an N-terminal aspartate residue of a peptidehaving a penultimate proline residue at the N-terminus. The peptide wasfurther conjugated to a solubilizing DNA linker which was attached to acyanine dye (Cy3B) for FRET and a biotin moiety for surfaceimmobilization.

In this experiment, the FRET peptide construct was loaded onto a glasschip having an array of sample wells, and collected light was filteredfirst by a green filter and then a red filter. Loading of the FRETpeptide construct was detected by measuring a signal passing throughboth the green and red filters. Terminal amino acid cleavage wasdetected when the signal was measurable only in the green filter, whichindicated that the red dye conjugated N-terminal amino acid from theFRET peptide construct was cleaved by yPIP. This detection pattern isillustrated in panel C. As shown, if both dyes are detectable before theaddition of yPIP, and only the green dye is visible after incubationwith yPIP, it can be reasonably concluded this change in detectionpattern is due to cleavage of the peptide and not photobleaching or lossof the peptide as a whole. Additionally, an increase in fluorescencefrom the lone green dye would be expected, as its emissions are nolonger absorbed by the red dye.

Following loading of the FRET peptide construct onto the chip, which hadbeen modified by surface passivation using phosphonic acid and silane,yPIP was introduced and images were obtained at several time points. Toassess the overall cleaving trend, the ratio of (green)/(green+red) wascomputed for each experiment. This ratio increases with the extent ofcleaving that occurs. FIG. 18B is a plot of FRET emission ratio acrossall apertures at different time points of incubation with yPIP. Asshown, the green dye contribution to the ratio of fluorescence emissionsincreases over time during incubation with yPIP, indicating that moreN-terminal aspartate residues have been cut, leaving behind thetruncated peptide with just the green dye.

Cutting efficiency was then evaluated at different time points bydetermining at which time points dye fluorescence was observed. This wasdone with simple thresholding—e.g., if the average dye emission signalwas >2.5 during excitation, the dyes were determined to be present (wheneach corresponding filter was applied). Apertures exhibiting cuttingwould then display both green and red dyes during the loading phase ofthe experiment, but only green dye at time points exposed to yPIP. Asshown in FIG. 18C, progressively more cutting was observed as the chipwas exposed to longer incubation times with yPIP. Example signal tracesshowing cutting displayed at each of the three yPIP-treated time pointsare shown in FIG. 18D.

Additional experiments were performed with yPIP and other peptidasesusing chips that had been modified by surface passivation using dextran,which produced similar results showing an increase in terminal aminoacid cleavage over time following introduction of peptidase onto chips.FIG. 18E is a plot of FRET emission ratio across loaded apertures atdifferent time points of incubation with yPIP. FIG. 18F is a plot ofFRET emission ratio across loaded apertures at different time points ofincubation with an aminopeptidase. Overall, the experiments heredemonstrate that N-terminal amino acid cleavage is detectable inreal-time at the single molecule level using different exopeptidases anddifferent labeling strategies.

Example 5. Terminal Amino Acid Discrimination by Labeled AffinityReagent

An adaptor protein involved in proteolytic pathways was identified as apotential candidate for use as a labeled affinity reagent for detectingN-terminal aromatic residues. The adaptor protein, ClpS2 from anα-proteobacterium (A. tumefaciens), was expressed and labeled at anexposed cysteine residue. FIG. 19A shows a crystal structure of theClpS2 protein, with the exposed cysteine residue shown as sticks. Theexposed cysteine residue was labeled with a rhodamine dye (ATTO 532).

Peptides having different N-terminal aromatic residues were prepared totest whether the labeled ClpS2 was capable of N-terminal amino aciddiscrimination at the single molecule level. Example single moleculeintensity traces from these experiments are shown in FIG. 19B. As shown,the signal traces demonstrate residue-specific on-off binding patternscorresponding to the labeled affinity reagent reversibly binding theN-terminus of peptides having either: an N-terminal phenylalanineresidue (F, top signal trace), an N-terminal tyrosine residue (Y, middlesignal trace), or an N-terminal tryptophan residue (W, bottom signaltrace).

Further analyses of the single molecule trajectories were carried out,with the results shown in FIGS. 19C-19E. FIG. 19C is a plot showingdiscriminant pulse durations (time duration of signal peaks) among thethree N-terminal residues when reversibly bound by labeled ClpS2. FIG.19D is a plot showing discriminant interpulse durations (time durationbetween signal pulses) among the three N-terminal residues. FIG. 19Eshows plots which further illustrate the discriminant pulse durationsamong phenylalanine, tyrosine, and tryptophan at peptide N-termini. Meanpulse duration for the different N-terminal residues is visualized byhistograms (A)-(B) and layered histogram (C).

Another adaptor protein, ClpS from Thermosynochoccus elongatus (teClpS)was evaluated for use as a labeled affinity reagent for leucinerecognition. The data obtained from dwell time analysis, shown in FIGS.19F-19H, demonstrated that the labeled teClpS protein producesdetectable binding interactions with a terminal leucine residue ofpolypeptides with a mean pulse duration of 0.71 seconds. The amino acidsequence of the teClpS protein used in these experiments is shown inTable 1.

Similar experiments were carried out to evaluate A. tumefaciens ClpS1and S. elongatus ClpS2 as potential reagents for leucine recognition,and GID4 as a potential reagent for proline recognition. FIG. 19I showsexample results from dwell time analysis which showed differentiablerecognition of phenylalanine, leucine, tryptophan, and tyrosine by A.tumefaciens ClpS1. FIG. 19J shows example results from dwell timeanalysis demonstrating leucine recognition by S. elongatus ClpS2. FIGS.19K-19L show example results from dwell time analysis demonstratingproline recognition by GID4.

Example 6. Polypeptide Sequencing by Recognition During Degradation

Experiments were conducted to evaluate peptide sequencing by N-terminalamino acid recognition during an ongoing degradation reaction. Exampleresults from these experiments are shown in FIGS. 20A-20D, which showsingle molecule intensity traces obtained over two independentpolypeptide sequencing reactions conducted in real-time using a labeledClpS2 protein and an aminopeptidase in the same reaction mixture. Ineach reaction, a polypeptide of sequence YAAWAAFADDDWK (SEQ ID NO: 78)was immobilized to a chip surface through the C-terminal lysine residueby loading the peptide composition (10 pM) onto chips for 20 minutes,and the immobilized peptide was monitored in the presence of a labeledaffinity reagent (ATTO 542-labeled A. Tumefaciens ClpS2-V1 at 500 nM)and an aminopeptidase cleaving reagent (VPr at 8 μM).

FIGS. 20A and 20C show signal trace data for two different sequencingruns, with the top panel (panel 1 in FIG. 20A, panel 2 in FIG. 20C)showing a full trace, and the bottom panels (Y, W, F) showing zoomed-inregions corresponding to each of the highlighted regions in the fulltrace. FIGS. 20B and 20D show pulse duration statistics in histogramsfor the trace data of the corresponding panels as labeled in FIGS. 20Aand 20C, respectively. As shown in the full signal trace of eachsequencing run (panels 1, 2), three separate time intervals of signalpulses were observed over the course of the reaction. As highlighted bythe zoomed-in regions (panels Y, W, F), the three intervals are visuallydistinguishable from one another based on an observable difference inpattern of signal pulses.

To further analyze the signal pulse data, pulse duration statistics weredetermined for each time interval (FIGS. 20B and 20D). The differencesin pulse duration distribution were determined to correspond to thoseobserved for these amino acids individually in steady-state on-chipbinding assays with ClpS2, and the signal pulse information wasphenotypically consistent between intervals from sequencing runs and theindividual amino acid binding assays.

As confirmed by the analysis of signal pulse information, the three timeintervals of signal pulses observed over the progression of eachsequencing run correspond to recognition patterns of Y, W, and F,respectively (panels 1, 2). The intervening time period between signalpulse patterns is due to the selectivity of ClpS2-V1, which does notbind to N-terminal alanine residues. As illustrated by the full signaltrace, the first interval corresponds to Y recognition, which isfollowed by a pause as VPr peptidase cuts Y and two alanine residues,followed by the second interval corresponding to W recognition, which isfollowed by another pause as VPr peptidase cuts W and two alanineresidues, and finally the third interval corresponding to F recognitionbefore VPr peptidase cuts off the F and stops at the remaining ADDDWKpeptide. These results show that pulse duration information, which wasobtained by terminal amino acid recognition during an ongoingdegradation reaction, can be used to determine characteristic patternsthat discriminate between different types of terminal amino acids.

Example 7. Terminal Amino Acid Identification and Cleavage by LabeledExopeptidase

Studies were performed to investigate the potential for a single reagentthat is capable of both identifying a terminal amino acid of a peptideand cleaving the terminal amino acid from the peptide. As a singlereagent, an exopeptidase must be able to bind to the peptide whileretaining cleavage activity toward a terminal residue. Accordingly, aninitial approach employing traditional labeling strategies was carriedout by targeting the native surface-exposed amino acids of differentexopeptidases. In these experiments, surface-exposed cysteine (—SH) orlysine (—NH₂) residues were labeled with fluorescent dyes, which provedto be a robust methodology for exopeptidase labeling. In certain cases,however, this approach produced a heterogeneous population of proteinsthat are labeled with one or more dyes.

In order to more precisely control where labeling occurs onexopeptidases and ensure that each exopeptidase molecule is labeled witha single fluorescent dye (as well as eliminate off-target reactivity ofthe dye), a new labeling strategy was investigated. In theseexperiments, labeled exopeptidases were prepared using a site-specificlabeling strategy in which an unnatural amino acid containing a reactivefunctional group is introduced into the exopeptidase (see, e.g., Chin,J. W., et al. J Am Chem Soc. 2002 Aug. 7; 124(31):9026-9027).

The proline iminopeptidase from Yersinia pestis (yPIP) was modified bymutation of a lysine residue at position 287 to a residue having apara-azidophenylalanine (pAzF) side chain. FIG. 21A shows a crystalstructure of yPIP, with the mutation indicated by the chemical structureof pAzF shown with the K287 sidechain shown as sticks. This mutationsite was selected based on the stability provided by the alpha helix atthis position and to ensure that the new azido functional group issolvent exposed.

A pEVOL plasmid containing the mutant amino tRNA synthetase and themutant tRNA necessary to incorporate pAzF into the amino acid chain wasobtained. The amber stop codon (TAG), which is necessary for thespecific incorporation of pAzF, was then introduced into the cDNA usingthe QuickChange II mutagenesis kit. The cDNA was then sequenced and theTAG codon position was confirmed. This was followed by co-transfectionof both the pET21b+ plasmid containing the yPIP amber mutant and thepEVOL plasmid containing the cellular machinery to charge the tRNA forthe amber codon with pAzF. The co-transfected cells were then grown to0.8 ODU, induced with 0.02% arabinose and 1 mM IPTG in the presence of 2mM pAzF in 2 L of LB, and harvested using chemolysis. Purification wascarried out using a 5 mL affinity chromatography column, and the proteinwas eluted in 100 mM imidazole. The resulting protein was then dialyzedand concentrated into 50 mM HEPES pH 8.0 and 0.5 M KCl, aliquoted, andflash frozen prior to storage at −20° C.

To confirm the presence of the azido group in the purified protein,DBCO-Cy3 (2 mM) was reacted with the pAzF-yPIP variant (220 μM)(Reaction Conditions: 50 mM HEPES pH 8.0, 0.5 mM KCl, 20% DMSO; 10 hoursat 37° C., 48 hours at room temperature). The protein reaction productwas purified by size-exclusion chromatography, and it was determinedthat the resulting protein was 100% labeled with the azide-reactiveDBCO-Cy3 reagent (FIG. 21B), indicating robust incorporation of theunnatural amino acid.

Protein labeling and purity of the final product was confirmed bySDS-PAGE analysis of the unlabeled and labeled pAzF variant. FIG. 21Cshows a picture of SDS-PAGE gel confirming Cy3-labeling of pAzF-yPIP(overexposed image of gel shown in FIG. 21D to show ladder). FIG. 21Eshows a picture of Coomassie-stained gel confirming that both dye andprotein co-migrate and are pure.

The dye-labeled pAzF-yPIP variant was used in an activity assay toconfirm that the enzyme was still active after labeling andpurification. As shown in FIG. 21F, Cy3-pAzF-yPIP was able to hydrolyze100% of the peptide substrate in 1 hour using 1000-fold excesssubstrate, as measured by HPLC. These experiments demonstrate amethodology which allows site-specific modification and labeling of anexopeptidase with minimal perturbation of the native proteinstructure/function.

Example 8. Recognition of Modified Amino Acids in Polypeptide Sequencing

Experiments were performed to evaluate recognition of amino acidscontaining specific post-translation modifications. A triple-mutantvariant (TBV, S10A, K15L) of the Src Homology 2 (SH2) domain from Fyn, atyrosine kinase, was tested as a potential recognition molecule forphosphorylated tyrosine residues in peptide sequencing. The variantprotein was immobilized to the bottom of sample wells, andsingle-molecule signal traces were collected upon addition of afluorescently-labeled peptide containing N-terminal phospho-tyrosine.Peptide binding by the immobilized protein was detected during theseexperiments, as shown by the representative traces in FIG. 22A. Pulseduration data collected during these experiments is shown in FIG. 22B(top, middle, and bottom plots corresponding to the top, middle, andbottom traces of FIG. 22A, respectively). Pulse duration and interpulseduration statistics are shown in FIG. 22C (top and bottom panels,respectively).

Control experiments were performed to confirm that the Fyn protein wasspecific for the phosphorylated tyrosine. The experiments were repeatedfor each of three different peptides: a first peptide containingN-terminal unmodified tyrosine (Y; FIG. 22D), a second peptidecontaining N-terminal and penultimate unmodified tyrosines (YY; FIG.22E), and a third peptide containing N-terminal phospho-serine (FIG.22F). As shown, binding was not detected with any of the peptides usedin the negative control experiments.

Example 9. Recognition of Penultimate Amino Acids in PolypeptideSequencing

Experiments were performed to determine the effects of penultimate aminoacids on pulse duration for A. Tumefaciens ClpS2-V1. Forty-ninedifferent fluorescently-labeled peptides were prepared containing uniquedipeptide sequences at the N-terminus, where the N-terminal amino acidwas F, W, or Y, and the penultimate position was one of the 20 naturalamino acids. For each experiment, ClpS2-V1 was immobilized at the bottomof sample wells, and single-molecule signal traces were collected for10-20 minutes upon addition of one of the fluorescently-labeledpeptides. Pulse duration data was collected for a minimum of 50 samplewells for each peptide.

FIG. 23 shows the median pulse duration for each of the 50 peptides,with data points grouped by penultimate amino acid (x-axis) andN-terminal amino acids represented with different symbols.

Example 10. Simultaneous Amino Acid Recognition with MultipleRecognition Molecules

Single-molecule peptide recognition experiments were performed todemonstrate terminal amino acid recognition of an immobilized peptide bymore than one labeled recognition molecule. Single peptide moleculescontaining N-terminal phenylalanine (FYPLPWPDDDY (SEQ ID NO: 79)) wereimmobilized in sample wells of a chip. Buffer containing 500 nM each ofatClpS1 (Agrobacterium tumifaciens ClpS1; sequence provided in Table 1)and atClpS2-V1 (Agrobacterium tumifaciens ClpS2 variant 1; sequenceprovided in Table 1) was added, where atClpS1 and atClpS2-V1 werelabeled with Cy3 and Cy3B, respectively. Since the intensity of Cy3B ishigher than Cy3, atClpS2-V1 binding events were readily distinguishablefrom atClpS1 binding events.

FIGS. 24A-24C shows the results of the experiments showingsingle-molecule peptide recognition with differentially labeledrecognition molecules. A representative trace is displayed in FIG. 24A.The pulse duration distributions were distinct for each binder (FIG.24B) and corresponded to their kinetic profiles as observed insingle-binder experiments. Mean pulse duration was 1.3 seconds foratClpS1 and 1.0 seconds for atClpS2-V1 (FIG. 24C). Pulse rate was alsodistinct: 8.1 pulses/min for atClpS1 and 14.1 pulses/min for atC1p2-V1(FIG. 24C). Thus, when more than one recognition molecule is includedfor dynamic recognition of immobilized peptides, the bindingcharacteristics of each recognition molecule (including pulse duration,interpulse duration, and pulse rate) can simultaneously provideinformation about peptide sequence.

Example 11. Enhancing Photostability with Recognition Molecule Linkers

Experiments were performed to evaluate the photostability of immobilizedpeptides during single-molecule sequencing. The dye-labeled atClpS2-V1described in Example 5 was added to sample wells containing immobilizedpeptide substrates in the presence of excitation light at 532 nm tomonitor recognition by emission from ATTO 532. A representative trace isshown in FIG. 25A. As shown in the top panel, recognition was observedto cease at approximately 600 seconds into the experiment. The bottompanel is a zoomed view showing signal pulses at approximately 180-430seconds into the reaction.

FIG. 25B shows a visualization of the crystal structure of the ClpS2protein used in these experiments. As shown, the cysteine residue thatserves as the dye conjugation site is approximately 2 nm from theterminal amino acid binding site. It was hypothesized that photodamageto the peptide was caused by proximity of the dye to the N-terminus ofpeptide during binding. To mitigate the potential photodamaging effectsof dye proximity, the ClpS2 protein was dye-labeled through a linkerthat increased distance between the dye and N-terminus of peptide bymore than 10 nm. The linker included streptavidin and a double-strandednucleic acid; the double-stranded nucleic acid was labeled with two Cy3Bdye molecules and attached to streptavidin through a bis-biotin moiety,and a ClpS2 protein was attached to each of the remaining two bindingsites on streptavidin through a biotin moiety. A representative traceusing this dye-shielded ClpS2 molecule is shown in FIG. 25C. As shown inthe top panel, recognition time was extended to approximately 6,000seconds into the experiment. The bottom panel is a zoomed view showingsignal pulses at approximately 750-930 seconds into the reaction.

A DNA-streptavidin recognition molecule was generated with a linkercontaining a double-stranded nucleic acid labeled with two Cy3B dyemolecules and attached to streptavidin through a bis-biotin moiety, anda single ClpS2 protein attached to the remaining two binding sites onstreptavidin through a bis-biotin moiety. This construct was used in asingle-molecule peptide sequencing reaction, and representative tracesfrom these experiments are shown in FIGS. 26A-26D.

The sequencing experiments described in example 6 were repeated, withthe reaction conditions changed as follows: the DNA-streptavidin ClpS2recognition molecule was used in combination with hTET amino acidcleaving reagent. A representative signal trace is shown in FIG. 27.

Example 12. Sequencing by Recognition During Degradation by MultipleExopeptidases

Experiments were performed to evaluate the use of multiple types ofexopeptidases with differential cleavage specificities in asingle-molecule peptide sequencing reaction mixture. Single peptidemolecules (YAAWAAFADDDWK (SEQ ID NO: 78)) were immobilized through aC-terminal lysine residue in sample wells of a chip. Buffer containingatClpS2-V1 for amino acid recognition and hTET for amino acid cleavagewas added. A representative trace is displayed in FIG. 28A, withexpanded views of pulse pattern regions shown in FIG. 28B.

An experiment was carried out to evaluate sequencing reactions in thepresence of two types of exopeptidases with differential specificities.Single peptide molecules (FYPLPWPDDDYK (SEQ ID NO: 80)) were immobilizedthrough a C-terminal lysine residue in sample wells of a chip. Buffercontaining atClpS2-V1 for amino acid recognition, and both hTET and yPIPfor amino acid cleavage was added. A representative trace is displayedin FIG. 28C, with expanded views of pulse pattern regions shown in FIG.28D. Additional representative traces from these reaction conditions areshown in FIG. 28E.

Further experiments were carried out to evaluate sequencing reactions inthe presence of two types of exopeptidases with differentialspecificities. Single peptide molecules (YPLPWPDDDYK (SEQ ID NO: 81))were immobilized through a C-terminal lysine residue in sample wells ofa chip. In one experiment, buffer containing atClpS2-V1 for amino acidrecognition, and both hTET and yPIP for amino acid cleavage was added. Arepresentative trace is displayed in FIG. 28F, with expanded views ofpulse pattern regions shown in FIG. 28G. Additional representativetraces from these reaction conditions are shown in FIG. 28H. In afurther experiment, buffer (50 mM MOPS, 60 mM KOAc, 200 μM Co(OAc)₂)containing atClpS2-V1 for amino acid recognition, and both PfuTET andyPIP for amino acid cleavage was added. A representative trace isdisplayed in FIG. 28I, with expanded views of pulse pattern regionsshown in FIG. 28J.

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of” “only one of” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03. It should be appreciatedthat embodiments described in this document using an open-endedtransitional phrase (e.g., “comprising”) are also contemplated, inalternative embodiments, as “consisting of” and “consisting essentiallyof” the feature described by the open-ended transitional phrase. Forexample, if the application describes “a composition comprising A andB,” the application also contemplates the alternative embodiments “acomposition consisting of A and B” and “a composition consistingessentially of A and B.”

Where ranges are given, endpoints are included. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof. Therecitation of an embodiment herein includes that embodiment as anysingle embodiment or in combination with any other embodiments orportions thereof.

1-14. (canceled)
 15. A method of polypeptide sequencing, the methodcomprising: contacting a single polypeptide molecule with one or moreterminal amino acid recognition molecules; and detecting a series ofsignal pulses indicative of association of the one or more terminalamino acid recognition molecules with successive amino acids exposed ata terminus of the single polypeptide while the single polypeptide isbeing degraded, thereby sequencing the single polypeptide molecule. 16.The method of claim 15, wherein association of the one or more terminalamino acid recognition molecules with each type of amino acid exposed atthe terminus produces a characteristic pattern in the series of signalpulses that is different from other types of amino acids exposed at theterminus.
 17. The method of claim 16, wherein the characteristic patterncomprises a portion of the series of signal pulses.
 18. The method ofclaim 17, wherein a signal pulse of the characteristic patterncorresponds to an individual association event between a terminal aminoacid recognition molecule and an amino acid exposed at the terminus. 19.The method of claim 18, wherein the signal pulse of the characteristicpattern comprises a pulse duration that is characteristic of adissociation rate of binding between the terminal amino acid recognitionmolecule and the amino acid exposed at the terminus.
 20. The method ofclaim 19, wherein each signal pulse of the characteristic pattern isseparated from another by an interpulse duration that is characteristicof an association rate of terminal amino acid recognition moleculebinding.
 21. The method of claim 16, wherein the characteristic patterncorresponds to a series of reversible terminal amino acid recognitionmolecule binding interactions with the amino acid exposed at theterminus of the single polypeptide molecule.
 22. The method of claim 21,wherein the series of reversible terminal amino acid recognitionmolecule binding interactions comprises a reversible formation of onebinary complex species at the terminus of the single polypeptidemolecule.
 23. (canceled)
 24. The method of claim 16, wherein thecharacteristic pattern is indicative of the amino acid exposed at theterminus of the single polypeptide molecule and an amino acid at acontiguous position. 25-26. (canceled)
 27. The method of claim 15,wherein sequencing comprises identifying at least a portion of all typesof successive amino acids exposed at the terminus of the singlepolypeptide while the single polypeptide is being degraded. 28-41.(canceled)
 42. The method of claim 15, wherein each of the one or moreterminal amino acid recognition molecules comprises a recognitionprotein or a recognition nucleic acid aptamer.
 43. The method of claim42, wherein the recognition protein is a degradation pathway protein, apeptidase, an antibody, an aminotransferase, a tRNA synthetase, or anSH2 domain-containing protein or fragment thereof. 44-52. (canceled) 53.The method of claim 15, wherein each of the one or more terminal aminoacid recognition molecules comprises a detectable label.
 54. The methodof claim 53, wherein the detectable label is a luminescent label or aconductivity label.
 55. The method of claim 15, wherein the singlepolypeptide molecule is immobilized to a surface. 56-60. (canceled) 61.The method of claim 15, wherein the single polypeptide molecule isdegraded by a cleaving reagent that removes one or more amino acids fromthe terminus of the single polypeptide molecule. 62-64. (canceled) 65.The method of claim 15, wherein the series of signal pulses is a seriesof real-time signal pulses.
 66. A system comprising: at least onehardware processor; and at least one non-transitory computer-readablestorage medium storing processor-executable instructions that, whenexecuted by the at least one hardware processor, cause the at least onehardware processor to perform the method of claim
 15. 67. At least onenon-transitory computer-readable storage medium storingprocessor-executable instructions that, when executed by at least onehardware processor, cause the at least one hardware processor to performthe method of claim
 15. 68-283. (canceled)
 284. The method of claim 16,wherein signal pulses of the characteristic pattern comprise a meanpulse duration of between about 1 millisecond and about 10 seconds. 285.The method of claim 284, wherein the mean pulse duration is betweenabout 10 milliseconds and about 100 milliseconds or between about 100milliseconds and about 500 milliseconds.
 286. The method of claim 16,wherein the characteristic pattern of one type of amino acid isdifferent from the characteristic pattern of another type of amino acidby a mean pulse duration of at least 10 milliseconds.
 287. The method ofclaim 286, wherein the characteristic pattern of one type of amino acidis different from the characteristic pattern of another type of aminoacid by a mean pulse duration of between about 10 milliseconds and about100 milliseconds or between about 100 milliseconds and about 10 seconds.288. The method of claim 61, wherein the reaction mixture comprises aterminal amino acid recognition molecule and the cleaving reagent in aratio of between about 10:1 and about 200:1.
 289. The method of claim61, wherein the reaction mixture comprises a terminal amino acidrecognition molecule and the cleaving reagent in a ratio of betweenabout 50:1 and about 150:1.